Benzopyrylium compounds

ABSTRACT

Compounds used as labels with properties comparable to known fluorescent compounds. The compounds are conjugated to proteins and nucleic acids for biological imaging and analysis. Synthesis of the compounds, formation and use of the conjugated compounds, and specific non-limiting examples of each are provided.

This application claims priority to U.S. Patent Application Ser. Nos.61/719,676 filed Oct. 29, 2012, and 61/693,918 filed Aug. 28, 2012, eachof which is expressly incorporated by reference in its entirety.

Compounds useful as labels with properties comparable to knownfluorescent compounds are disclosed. The compounds can be conjugated toproteins and nucleic acids for biological imaging and analysis.Synthesis of the compounds, formation and use of the conjugatedcompounds, and specific non-limiting examples of each are disclosed. Thecompounds are benzopyrylo-polymethine based compounds, abbreviated asbenzopyrylium compounds, that contain at least one ethylene glycol.

Organic fluorescent compounds, also referred to as dyes, are used assensitive detection reagents in biological assays.Benzopyrylo-polymethine based dyes are commercially available asresearch reagents.

Compounds that contain at least one ethylene glycol, diethylene glycol,or polyethylene glycol (i.e., (poly)ethylene glycol, PEG, or pegylatedbenzopyrylium derivatives), specifically sulfonamide derivatives andaryl sulfonamide derivatives of compounds containing abenzopyrylo-polymethine ring structure are disclosed. These are referredto as fluorescent pegylated benzopyrylo-polymethine compounds or dyes.Adding one or more aryl sulfonamide derivatives, as well as addingethylene glycol and/or polyethylene glycol (PEG) moieties to thebenzopyrylo-polymethine compound, e.g., at the sulfonamide nitrogen,results in enhanced hydrophilicity and other properties over those ofunsubstituted compounds. In one embodiment, the compounds optionallycontain a reactive group that may be used for covalent coupling tobiomolecules.

These compounds have enhanced fluorescence, water solubility, andbiocompatibility, and are used in compositions and methods asfluorescent probes in biological and other types of assays. Theirproperties compare favorably with negatively charged sulfonatederivatives.

Compounds that react with biomolecules (e.g., antigens, antibodies,DNA-segments with the corresponding complimentary species for measuringenzyme kinetics, receptor-ligand interactions, nucleic acidhybridization kinetics in vitro as well as in vivo, etc.), termed labelsor dyes, are useful for, e.g., pharmacological characterization ofreceptors and drugs, binding data, etc. Compounds such as xanthyliumsalts (U.S. Pat. No. 5,846,737) and/or cyanines (U.S. Pat. No.5,627,027) are used for such applications, but aggregate and formdimers, especially in aqueous solution, due to planarity of theirπ-system. Compounds that have insufficient hydrophilicity undergonon-specific interactions with various surfaces, resulting in problemswhen attempting purify the corresponding conjugate, and anunsatisfactory signal to noise ratio.

Efforts are directed to reducing undesirable properties by introducingsubstituents that increase the hydrophilicity of the compounds. Thechallenge was to create highly hydrophilic content polymethine-basedfluorescent markers with high extinction coefficients and a high degreeof photo and storage stability. These can be excited simply to emitfluorescent light by monochromatic (laser/laser diodes) or polychromaticlight (white light sources) in the ultraviolet (UV), visual, or nearinfrared (NIR) spectrum range, or can function as quenchers.

Symmetrical xanthylium salt (fluorescein and rhodamine) or polymethine(indocyanine) as claimed in, for example, U.S. Pat. No. 5,627,027 arenormally used. All these markers have the disadvantage that they tendtowards aggregation and dimerization owing to the planarity of thepi-electron system, especially in aqueous systems. Moreover,insufficiently hydrophilic markers show non-specific interactions withdifferent surfaces, resulting in problems with clearing thecorresponding conjugates and in an unsatisfactory signal/noise ratio. Tocircumvent these disadvantages, corresponding asymmetrical polymethineson the basis of benzopyran-2-ylide or benzo[b]pyran-4-ylide-compoundswere described in PCT/DE00/00802, PCT/DE01/01946, and U.S. Pat. No.6,924,372.

The inventive compounds and methods are improved by introducingadditional substituents that increased hydrophilicity of the compounds.Compounds that contain at least one ethylene glycol, diethylene glycol,or polyethylene glycol (i.e., (poly)ethylene glycol, PEG), or pegylatedbenzopyrylo-polymethine-based compounds for use in optical, and inparticular optical-fluorescent, determination and detection procedures,e.g., in medicine, the pharmaceutical industry and in bioscience,materials science, and environmental science, are disclosed. Thedisclosed compounds are useful as labels in optical, especiallyfluorescence optical, determination and detection methods. The compoundshave high hydrophilicity, high molar absorbance, high photo-stability,and high storage stability. These compounds can be excited bymonochromatic (e.g., lasers, laser diodes) or polychromatic (e.g., whitelight sources) light in the UV, visible, and NIR spectral region togenerate emission of fluorescence light.

Typical application methods are based on the reaction of the compoundswith biomolecules such as proteins (e.g., antigens, antibodies, etc.),DNA and/or RNA segments, etc. with the corresponding complimentaryspecies. Thus, among other embodiments, the compounds are useful tomeasure enzyme kinetics, receptor-ligand interactions, and nucleic acidhybridization kinetics in vitro and/or in vivo. The compounds are usefulfor the pharmacological characterization of receptors and/or drugs.Applications include, but are not limited to, uses in medicine,pharmacy, biological sciences, materials sciences, environmentalcontrol, detection of organic and inorganic micro samples occurring innature, etc.

FIG. 1 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 2 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 3 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 4 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 5 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 6 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 7 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 8 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 9 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 10 shows functional assay results with commercial dyes and theinventive compounds in one embodiment.

FIG. 11 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 12 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 13 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 14 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 15 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 16 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 17 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 18 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 19 shows immunofluorescence assay results with commercial dyes andthe inventive compounds in one embodiment.

FIG. 20 shows in vivo imaging and biodistribution of one inventivecompound 675-P (V03-07005).

FIG. 21 shows ex vivo accumulation in an in vivo assay of one inventivecompound 675-P (V03-07005).

FIG. 22 shows in vivo clearance of one inventive compound 675-P(V03-07005).

FIGS. 23A-E show histology of one inventive compound 675-P (V03-07005)in an in vivo assay.

FIG. 24 shows in vivo imaging and biodistribution of a commercialcompound (DyLight 680).

FIG. 25 shows ex vivo accumulation in an in vivo assay of a commercialcompound (DyLight 680).

FIG. 26 shows in vivo clearance of a commercial compound (DyLight 680).

FIGS. 27A-E show histology in an in vivo assay of a commercial compound(DyLight 680).

The following nomenclature is used to describe various embodiments: 682Compound X, where X indicates position(s) on general formula I or IIwhich contains PEG group(s). For example, in one embodiment, 682Compound 2/3 contains at least ethylene glycol, diethylene glycol, or(poly)ethylene glycol at each of R2 and R3.

In one embodiment, the compounds have at least one ethylene glycolgroup, diethylene glycol group, or ethylene glycol polymer (i.e.,poly(ethylene) glycol, abbreviated as PEG) in any position of thecompound, and a function for conjugating the compound to a biomoleculein any position of the compound. In one embodiment, the compound has, inany position of the compound, at least one sulfo and/or sulfoalkyl. Inone embodiment, the compound has, in any position of the compound, asulfonamide and/or carboxamide group comprising an ethylene glycol groupor an ethylene glycol polymer (i.e., poly(ethylene) glycol, abbreviatedas PEG), either directly or indirectly attached to the compound.Indirect attachment indicates use of a linker, direct attachmentindicates lack of such a linker. A linker can be any moiety.

In one embodiment, the compound is general formula I

or general formula II

where each of R¹, R², R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is the same ordifferent and is independently selected from H, SO₃, Z, L-Z, a PEG groupP-L-Z where P is an ethylene glycol group, a diethylene glycol group, ora polyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L-SO₂NH—P-L-Z, a carboxamide group -L-CONH—P-L-Z, hydrogen,alkyl-, tert-alkyl, aryl-, carboxyaryl-, dicarboxyaryl, heteroaryl-,cycloalkyl-, heterocycloalkyl-, alkyloxy-, alkylmercapto- with alkyl andcycloalkyl including olefin linkage residues, aryloxy-, arylmercapto-,heteroaryloxy-, heteroarylmercapto-, hydroxy-, nitro-, a carboxylicacid, an amino group, or cyano residues; where L is a divalent linear(—(CH₂)_(t)—, t=0 to 15), branched, or cyclic alkane group that can besubstituted by at least one atom of oxygen, nitrogen, substitutednitrogen, and/or sulfur; where Z is H, CH₃, alkyl group, sulfoalkyl,heteroalkyl group, NH₂, —COO⁻, —COOH, —COSH, CO—NH—NH₂, —COF,

—COCl, —COBr, —COI, —COO-Su (succinimidyl/sulfosuccinimidyl), —COO-STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO-Su, —CONR′-L-COO-STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, or—CONR′-L-NH—CO—CH₂—I; each of R′ and R″ is selected from H, aliphaticgroup, or heteroaliphatic group, and the biomolecule is a protein,peptide, antibody, nucleotide, oligonucleotide, biotin, or hapten;

each of R¹⁰, R¹³, and R¹⁴ is the same or different and is independentlyselected from aliphatic, heteroaliphatic, sulfoalkyl group, carboxyalkylgroup, heteroaliphatic with terminal SO₃, Z, L-Z, PEG group P-L-Z whereP is an ethylene glycol group, a diethylene glycol group, or apolyethylene glycol group where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L-SO₂NH—P-L-Z, or a caboxamide group -L-CONH—P-L-Z;

each of R⁷ and R⁹ is the same or different and is independentlyhydrogen, aliphatic group, heteroaliphatic group, or PEG group P-L-Zwhere P is selected from an ethylene glycol group, a diethylene glycolgroup, or a polyethylene glycol group where the polyethylene glycolgroup is (CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive; or R⁷and R⁹ together form a cyclic structure where R³ and R⁴ are joined usinga divalent structural element selected from —(CH₂)_(q)—,—(CH₂)_(q)O(CH₂)_(q′)—,

—(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—, —OCH═CH— where each of q andq′ is the same or different and is a integer from 1 to 6 inclusive; and

R⁸ is selected from hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, and PEG group P-L-Z where P is selected from an ethylene glycolgroup, a diethylene glycol group, or a polyethylene glycol group, wherethe polyethylene glycol group is (CH₂CH₂O)_(s), where s is an integerfrom 3-6 inclusive;

each of R¹ and R², R² and R³, R³ and R⁴, R⁵ and R⁶, R⁵ and R⁸, R⁹ andR¹⁰, R¹¹ and R¹², or R¹² and R¹³ may form one or more aliphatic,heteroaliphatic, or aromatic rings, and where the resultant ring(s) isoptionally substituted by at least one alkyl-, sulfoalkyl, tert-alkyl,aryl-, carboxyaryl-, dicarboxyaryl, heteroaryl-, cycloalkyl-,heterocycloalkyl-, alkyloxy-, alkylmercapto- with alkyl and cycloalkylincluding olefin linkage residues, aryloxy-, arylmercapto-,heteroaryloxy-, heteroarylmercapto-, hydroxy-, nitro-, sulfonic acid, acarboxylic acid, an amino group, or cyano residues;

at least one of R¹-R¹⁴ may constitute an additional solubilizing agent,or ionizing or ionized substituent, besides PEG, such as SO₃ ⁻, PO₃ ²⁻,CO₂H, OH, NR₃ ⁺, cyclodextrins or sugars, which provide hydrophiliccharacteristics of dyes; these substituents may also be linked to theactual basic chromophore by an aliphatic or heteroaliphatic or cyclicalspacer group, with the proviso that at least one of R¹-R¹⁴ contains aPEG group; and n is 0, 1, 2, or 3.

In one embodiment, PEG group P is selected from —CH₂—CH₂—O—CH₃ (ethyleneglycol with terminal methyl), —CH₂—CH₂—O—CH₂—CH₂—O—CH₃ (diethyleneglycol with terminal methyl), —CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃(polyethylene glycol (3) with terminal methyl),—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃ (polyethylene glycol (4)with terminal methyl),—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃ (polyethyleneglycol (5) with terminal methyl), andCH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃(polyethylene glycol (6) with terminal methyl). In one embodiment, PEGgroup P may be either uncapped, e.g., lack a terminal methyl, or may becapped with an atom or group other than methyl. In one embodiment, PEGgroup P terminates with a Z group, where Z is defined above and includesH, CH₃, a CH₃ group, an alkyl group, or a heteroalkyl group.

In one embodiment, the compound is general formula Ia, Ib, Ic, Id, Ie,If, Ig, Ih or general formula IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, asshown below:

where each of R¹-R¹⁴ is as defined above, X is selected from —OH, —SH,—NH₂, —NH—NH₂, —F, —CI, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, or—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge(s); o is an integer from 0 to12 inclusive; p is an integer from 1 to 6 inclusive; and n is 0, 1, 2,or 3.

In one embodiment, the compound is general formula Ia where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula Ib where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula Ic where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula Id where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula Ie where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In one embodiment, the compound is general formula If where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In one embodiment, the compound is general formula Ig where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In one embodiment, the compound is general formula Ih where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In one embodiment, the compound is general formula IIa where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula IIb where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula IIc where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula IId where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R13 and R14 are independently selected fromalkyl, sulfoalkyl, or a PEG group P-L-Z, where P, L, and Z are definedabove; and R1, R4, R7, R8, R9, and R12 are H. In one embodiment, R2and/or R3 is sulfopropyl. In one embodiment, R5 is methyl. In oneembodiment, R13 and/or R14 is sulfopropyl.

In one embodiment, the compound is general formula IIe where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is either sulfonic acid,carboxylic acid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEGgroup P-L-Z, where P, L, and Z are defined above; R14 is alkyl; and R1,R4, R7, R8, R9, and R12 are H. In one embodiment, R2 and/or R3 issulfopropyl. In one embodiment, R5 is methyl. In one embodiment, R10 issulfopropyl. In one embodiment, R14 is methyl.

In one embodiment, the compound is general formula IIf where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In one embodiment, the compound is general formula IIg where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In one embodiment, the compound is general formula IIh where R2 and R3are independently selected from sulfoalkyl or a PEG group P-L-Z, whereP, L, and Z are defined above; R5 is alkyl; R6 is either t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z,where P, L, and Z are defined above; R14 is alkyl; and R1, R4, R7, R8,R9, and R12 are H. In one embodiment, R2 and/or R3 is sulfopropyl. Inone embodiment, R5 is methyl. In one embodiment, R10 is sulfopropyl. Inone embodiment, R14 is methyl.

In embodiments, and as described above, R2 and/or R3, in conjunctionwith other position(s), may form additional ring(s). In one embodiment,the benzopyrylium group of the described compound comprises additionalring(s). For example, in one embodiment, as shown in the benzopyryliumportion of general formula II below, R2 and R3 each form an additional6-membered ring, forming an N-bridged compound. In one embodiment, thecompound is an N-bridged compound where R5 is alkyl; R6 is eithert-butyl or an unsubstituted or substituted phenyl; and R7, R8, and R9are H. In one embodiment, R5 is methyl. In one embodiment, the N-bridgedcompound is according to general formula I.

In one embodiment, as shown in the benzopyrylium portion of generalformula II below, R3 forms an additional, substituted 6-membered ring,forming an N-bridged compound. In one embodiment, the compound is anN-bridged compound where R2 is sulfoalkyl or a PEG group P-L-Z, where P,L, and Z are defined above; R3′ is sulfonic acid, carboxylic acid, or anamino group; R5 is alkyl; R6 is either t-butyl or an unsubstituted orsubstituted phenyl; and R7, R8, and R9 are H. In one embodiment, R2 issulfopropyl. In one embodiment, R3′ is sulfonic acid. In one embodiment,R5 is methyl. In one embodiment, the N-bridged compound is according togeneral formula I.

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(4-tert-butyl-7-((2-methoxyethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),shown below, which contains an ethylene glycol (PEG₁) bound to thebenzopyrylium by N, i.e., a methylated ethylene glycol, and —COOH atR13. The methyl group on the ethylene glycol/PEG prevents the terminal—OH from oxidation. Oxidation is known to occur, over time, on anunprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 682 Compound 2 (PEG₁), but one skilled in theart appreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is an NHS-ester of 682Compound 2 (PEG₁), shown below:

One non-limiting example of an activated 682 Compound 2 (PEG₁) is atetrafluorophenyl (TFP)-ester form of 682 Compound 2, shown below:

One non-limiting example of an activated 682 Compound 2 (PEG₁) is asulfotetrafluorophenyl (STP)-ester form of 682 Compound 2, shown below:

One non-limiting example of an activated 682 Compound 2 (PEG₁) is ahydrazide form of 682 Compound 2, shown below:

One non-limiting example of an activated 682 Compound 2 (PEG₁) is amaleimide form of 682 Compound 2, shown below:

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-methoxyethoxyl)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),shown below, which contains an diethylene glycol (PEG₂) bound to thebenzopyrylium by N, i.e., a methylated diethylene glycol, and —COOH atR13.

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2 (PEG₂), shown below:

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-(2-methoxyethoxyl)ethoxy)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),shown below, which contains a polyethylene glycol (PEG₃) bound to thebenzopyrylium by N, i.e., a methylated polyethylene glycol, and —COOH atR13.

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2 (PEG₃), shown below:

In one embodiment, the compound is 682 Compound 2 (V17-03019)((E)-2-((E)-3-(4-tert-butyl-7-((3-sulfonatopropyl)(2,5,8,11-tetraoxatridecan-13-yl)amino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),shown below, which contains a polyethylene glycol (PEG₄) bound to thebenzopyrylium by N, i.e., a methylated polyethylene glycol, and

—COOH at R13.

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(7-(2,5,8,11,14-pentaoxahexadecan-16-yl(3-sulfonatopropyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),shown below, which contains a polyethylene glycol (PEG₅) bound to thebenzopyrylium by N, i.e., a methylated polyethylene glycol, and

—COOH at R13.

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2 (PEG₅), shown below:

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(7-(2,5,8,11,14,17-hexaoxanonadecan-19-yl(3-sulfonatopropyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),shown below, which contains a polyethylene glycol (PEG₆) bound to thebenzopyrylium by N, i.e., a methylated polyethylene glycol, and

—COOH at R13.

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2 (PEG₆), shown below:

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-methoxyethoxyl)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) bound to the benzopyrylium by N, i.e., amethylated ethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₁), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-methoxyethoxyl)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains adiethylene glycol (PEG₂) bound to the benzopyrylium by N, i.e., amethylated diethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₂), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₃) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₃), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2 (V08-16072)((E)-2-((E)-3-(4-tert-butyl-7-((3-sulfonatopropyl)(2,5,8,11-tetraoxatridecan-13-yl)amino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₄) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₄), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(7-(2,5,8,11,14-pentaoxahexadecan-16-yl(3-sulfonatopropyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₅) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₅), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2((E)-2-((E)-3-(7-(2,5,8,11,14,17-hexaoxanonadecan-19-yl(3-sulfonatopropyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₆) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₆), shown below

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2 (PEG₁), shown below:

or according to general formula II shown below

One non-limiting example of an activated 682 Compound 2 (PEG₁) accordingto general formula I is a tetrafluorophenyl (TFP)-ester form of 682Compound 2, shown below:

or according to general formula II shown below

One non-limiting example of an activated 682 Compound 2 (PEG₁) accordingto general formula I is a sulfotetrafluorophenyl (STP)-ester form of 682Compound 2, shown below:

or according to general formula II shown below

One non-limiting example of an activated 682 Compound 2 (PEG₁) accordingto general formula I is a hydrazide form of 682 Compound 2, shown below:

or according to general formula II shown below

One non-limiting example of an activated 682 Compound 2 (PEG₁) accordingto general formula I is a maleimide form of 682 Compound 2, shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(2-(2-carboxyethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) on the indole N, terminating with —COOH, at R10:

or according to general formula II shown below:

In one embodiment, the compound is 682 Compound 10, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onthe indole N, terminating with —OH, at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 10 (PEG₁), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(2-(2-(2-carboxyethoxy)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains andiethylene glycol (PEG₂) on the indole N, terminating with a —COOH, atR10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10, according to generalformula I and shown below, which contains an diethylene glycol (PEG₂) onthe indole N, terminating with a —OH, at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 10 (PEG₂), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains anpolyethylene glycol (PEG₃) on the indole N, terminating with a —COOH, atR10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₃)on the indole N, terminating with a —OH, at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 10 (PEG₃), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(14-carboxy-3,6,9,12-tetraoxatetradecyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains anpolyethylene glycol (PEG₄) on the indole N, terminating with a —COOH, atR10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₄)on the indole N, terminating with —OH, at

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 10 (PEG₄), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(17-carboxy-3,6,9,12,15-pentaoxaheptadecyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₅) on the indole N, terminating with a —COOH, atR10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₅)on the indole N, terminating with —OH, at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 10 (PEG₅), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(20-carboxy-3,6,9,12,15,18-hexaoxaicosyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains anpolyethylene glycol (PEG₆) on the indole N, terminating with —COOH, atR10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 10, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₆)on the indole N, terminating with —OH, at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 10 (PEG₆), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/10((E)-2-((E)-3-(4-tert-butyl-7-((2-methoxyethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(2-(2-carboxyethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) bound to the benzopyrylium by N, i.e., amethylated ethylene glycol, an ethylene glycol (PEG₁) on the indole N,and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/10 (PEG₁), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/10((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-methoxyethoxyl)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(2-(2-(2-carboxyethoxy)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains adiethylene glycol (PEG₂) bound to the benzopyrylium by N, i.e., amethylated diethylene glycol, a diethylene glycol (PEG₂) on the indoleN, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/10 (PEG₂), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/10((E)-2-((E)-3-(4-tert-butyl-7-((2-(2-(2-methoxyethoxyl)ethoxy)ethyl)(3-sulfonatopropyl)amino)chromenylium-2-yl)allylidene)-1-(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₃) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, a polyethylene glycol (PEG₃) on theindole N, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/10 (PEG₃), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/10 (V13-06190)((E)-2-((E)-3-(4-tert-butyl-7-((3-sulfonatopropyl)(2,5,8,11-tetraoxatridecan-13-yl)amino)chromenylium-2-yl)allylidene)-1-(14-carboxy-3,6,9,12-tetraoxatetradecyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₄) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, a polyethylene glycol (PEG₄) on theindole N, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/10 (PEG₄), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/10((E)-2-((E)-3-(7-(2,5,8,11,14-pentaoxahexadecan-16-yl(3-sulfonatopropyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(17-carboxy-3,6,9,12,15-pentaoxaheptadecyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₅) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, a polyethylene glycol (PEG₅) on theindole N, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/10 (PEG₅), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/10((E)-2-((E)-3-(7-(2,5,8,11,14,17-hexaoxanonadecan-19-yl(3-sulfonatopropyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(20-carboxy-3,6,9,12,15,18-hexaoxaicosyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₆) bound to the benzopyrylium by N, i.e., amethylated polyethylene glycol, and a polyethylene glycol (PEG₆) on theindole N, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/10 (PEG₆), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)methylene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains two ethyleneglycols (PEG₁) bound to the benzopyrylium by N, i.e., a methylatedethylene glycol, and —COOH at R13. The methyl group on the ethyleneglycol/PEG prevents the terminal —OH from oxidation, with a monomethinelinker connecting the benzopyrylium with the indole group. Oxidation isknown to occur, over time, on an unprotected PEG terminus. Adding amethyl ether provides this protection, and prevents reaction withelectrophilic reactive groups:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains two ethyleneglycols (PEG₁) bound to the benzopyrylium by N, i.e., a methylatedethylene glycol, —COOH at R13, and with a trimethine linker connectingthe benzopyrylium with the indole group:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E)-5-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)penta-2,4-dienylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains two ethyleneglycols (PEG₁) bound to the benzopyrylium by N, i.e., a methylatedethylene glycol, and

—COOH at R13:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E,6E)-7-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)hepta-2,4,6-trienylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains two ethyleneglycols (PEG₁) bound to the benzopyrylium by N, i.e., a methylatedethylene glycol, and —COOH at R13:

or according to general formula II shown below

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 682 Compound 2/3 (PEG₁), but one skilled inthe art appreciates that activation is not limited to these examples.One non-limiting example of an activated compound is an NHS-ester of 682Compound 2/3 (PEG₁) according to general formula I, shown below:

or according to general formula II shown below:

One non-limiting example of an activated 682 Compound 2/3 (PEG₁) is atetrafluorophenyl (TFP)-ester form of 682 Compound 2/3, shown below:

One non-limiting example of an activated 682 Compound 2/3 (PEG₁) is asulfotetrafluorophenyl (STP)-ester form of 682 Compound 2/3, shown below

One non-limiting example of an activated 682 Compound 2/3 (PEG₁) is ahydrazide form of 682 Compound 2/3, shown below

One non-limiting example of an activated 682 Compound 2/3 (PEG₁) is amaleimide form of 682 Compound 2/3, shown below

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2/3 (PEG₁), shown below

One non-limiting example of an activated compound is an NHS-ester of 682Compound 2/3 (PEG₁), shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-methoxyethoxyl)ethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains twodiethylene glycols (PEG₂) bound to the benzopyrylium by N, positions R2and R3, i.e., a methylated diethylene glycol, and —COOH at R13:

or according to general formula II shown below

One non-limiting example of an activated compound is the NHS-ester of682 Compound 2/3 (PEG₂) according to general formula I, shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains twopolyethylene glycols (PEG₃) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R13:

or according to general formula II shown below

One non-limiting example of an activated compound is the NHS-ester of682 Compound 2/3 (PEG₃) according to general formula I, shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3 (V03-07005)((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11-tetraoxatridecan-13-ylamino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains twopolyethylene glycols (PEG₄) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R13:

or according to general formula II shown below

One non-limiting example of an activated compound is the NHS-ester of682 Compound 2/3 (PEG₄) according to general formula I, shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11,14-pentaoxahexadecan-16-ylamino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains twopolyethylene glycols (PEG₅) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R13:

or according to general formula II shown below

One non-limiting example of an activated compound is the NHS-ester of682 Compound 2/3 (PEG₅) according to general formula I, shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11,14,17-hexaoxanonadecan-19-ylamino)chromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate)according to general formula I, shown below, which contains twopolyethylene glycols (PEG₆) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R13:

or according to general formula II shown below

One non-limiting example of an activated compound is the NHS-ester of682 Compound 2/3 (PEG₆) according to general formula I, shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twoethylene glycols (PEG₁) bound to the benzopyrylium by N, positions R2and R3, i.e., a methylated ethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₁), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-methoxyethoxyl)ethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twodiethylene glycols (PEG₂) bound to the benzopyrylium by N, positions R2and R3, i.e., a methylated diethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₂), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₃) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₃), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11-tetraoxatridecan-13-ylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₄) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₄), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11,14-pentaoxahexadecan-16-ylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₅) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₅), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11,14,17-hexaoxanonadecan-19-ylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₆) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₆), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)methylene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) bound to the benzopyrylium by N, i.e., amethylated ethylene glycol, with a monomethine linker connecting thebenzopyrylium with the indole group, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) bound to the benzopyrylium by N, i.e., amethylated ethylene glycol, with a trimethine linker connecting thebenzopyrylium with the indole group, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E)-5-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)penta-2,4-dienylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) bound to the benzopyrylium by N, i.e., amethylated ethylene glycol, with a pentamethine linker connecting thebenzopyrylium with the indole group, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E,6E)-7-(7-(bis(2-methoxyethyl)amino)-4-tert-butylchromenylium-2-yl)hepta-2,4,6-trienylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains anethylene glycol (PEG₁) bound to the benzopyrylium by N, i.e., amethylated ethylene glycol, with a heptamethine linker connecting thebenzopyrylium with the indole group, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₁), shown below:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₁), shown below:

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-methoxyethoxyl)ethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twodiethylene glycols (PEG₂) bound to the benzopyrylium by N, positions R2and R3, i.e., a methylated diethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₂), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)amino)-4-tert-butylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₃) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₃), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11-tetraoxatridecan-13-ylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₄) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₄), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11,14-pentaoxahexadecan-16-ylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₅) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₅), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(4-tert-butyl-7-(di2,5,8,11,14,17-hexaoxanonadecan-19-ylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₆) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is the NHS-ester of 682 Compound 2/3 (PEG₆), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a monomethinelinker connecting the benzopyrylium with the indole group, and

—COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a trimethinelinker connecting the benzopyrylium with the indole group, and —COOH atR10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E)-5-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)penta-2,4-dienylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a pentamethinelinker connecting the benzopyrylium with the indole group, and

—COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E,6E)-7-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)hepta-2,4,6-trienylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a heptamethinelinker connecting the benzopyrylium with the indole group, and

—COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₁), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-methoxyethoxyl)ethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₂) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₂), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-(2-(2-methoxyethoxyl)ethoxy)ethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₃) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₃), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-1-(5-carboxypentyl)-2-((E)-3-(7-(di2,5,8,11-tetraoxatridecan-13-ylamino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₄) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₄), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-1-(5-carboxypentyl)-2-((E)-3-(7-(di2,5,8,11,14-pentaoxahexadecan-16-ylamino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₅) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₅), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-1-(5-carboxypentyl)-2-((E)-3-(7-(di2,5,8,11,14,17-hexaoxanonadecan-19-ylamino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₆) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, and —COOH at R10:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₆), shown below:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a monomethinelinker connecting the benzopyrylium with the indole group, and —COOH atR13:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((E)-3-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)allylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a trimethinelinker connecting the benzopyrylium with the indole group, and —COOH atR13:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E)-5-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)penta-2,4-dienylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a pentamethinelinker connecting the benzopyrylium with the indole group, and —COOH atR13:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 2/3((E)-2-((2E,4E,6E)-7-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)hepta-2,4,6-trienylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains twopolyethylene glycols (PEG₁) bound to the benzopyrylium by N, positionsR2 and R3, i.e., a methylated polyethylene glycol, with a heptamethinelinker connecting the benzopyrylium with the indole group, and —COOH atR13:

or according to general formula II shown below

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2/3 (PEG₁), shown below:

In one embodiment, the compound is 682 Compound 1, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the benzopyrylium, position R1, i.e., amethylated ethylene glycol, via a sulfonamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 1, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the benzopyrylium, position R1, i.e., amethylated ethylene glycol, via a carboxamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 4, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the benzopyrylium, position R4, i.e., amethylated ethylene glycol, via a sulfonamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 4, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the benzopyrylium, position R4, i.e., amethylated ethylene glycol, via a carboxamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 11, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the indole, position R11, i.e., amethylated ethylene glycol, by a sulfonamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 11, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the indole, position R11, i.e., amethylated ethylene glycol, by a carboxamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 12, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the indole, position R12, i.e., amethylated ethylene glycol, by a sulfonamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 12, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) onan aromatic ring position of the indole, position R12, i.e., amethylated ethylene glycol, via a carboxamide linker, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, according to generalformula I and shown below, which contains an ethylene glycol (PEG₁) on aring position of the indole, position R13, i.e., a methylated ethyleneglycol, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, according to generalformula I and shown below, which contains an diethylene glycol (PEG₂) ona ring position of the indole, position R13, i.e., a methylateddiethylene glycol, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₃)on a ring position of the indole, position R13, i.e., a methylatedpolyethylene glycol, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₄)on a ring position of the indole, position R13, i.e., a methylatedpolyethylene glycol, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₅)on a ring position of the indole, position R13, i.e., a methylatedpolyethylene glycol, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, according to generalformula I and shown below, which contains an polyethylene glycol (PEG₆)on a ring position of the indole, position R13, i.e., a methylatedpolyethylene glycol, and —COOH at R10:

or according to general formula II shown below

In one embodiment, the compound is 682 Compound 13, shown below, whichcontains an ethylene glycol (PEG₁) on a N-ring position of the indole,position R13, i.e., a methylated ethylene glycol, via a sulfonamidelinker, and —COOH at R10.

In one embodiment, the compound is 682 Compound 13, shown below, whichcontains an ethylene glycol (PEG₁) on a N-ring position of the indole,position R13, i.e., a methylated ethylene glycol, via a carboxamidelinker, and —COOH at R10.

In one embodiment, the compound is 682 Compound 2/3/10, shown below,which contains two ethylene glycols (PEG₁) bound to the benzopyrylium byN, positions R2 and R3, i.e., a methylated ethylene glycol, and ethyleneglycol at R10 that terminates in —COOH.

In one embodiment, the compound is 682 Compound 2/3/10/13, shown below,which contains two ethylene glycols (PEG₁) bound to the benzopyrylium byN, positions R2 and R3, i.e., a methylated ethylene glycol, ethyleneglycol at R10 that terminates in —COOH, and ethylene glycol, i.e., amethylated ethylene glycol, at R13.

In one embodiment, the compound is 682 Compound 2((E)-2-((2E,4E,6E)-7-(7-(bis(2-methoxyethyl)amino)-3-methyl-4-phenylchromenylium-2-yl)hepta-2,4,6-trienylidene)-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR2, i.e., a methylated polyethylene glycol, sulfoalkyl at position R3,with a pentamethine linker connecting the benzopyrylium with the indolegroup, and —COOH at R13:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 2 (PEG₁), shown below:

In one embodiment, the compound is 682 Compound 3((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(2-methoxyethyl)amino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, with a trimethine linkerconnecting the benzopyrylium with the indole group, methyl at R13 andR14, t-butyl at R6, and

—COOH at R10 (V08-17084):

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 3 (PEG₁), shown below:

In one embodiment, the compound is 682 Compound 3((E)-2-((E)-3-(4-tert-butyl-7-(ethyl(2,5,8,11-tetraoxatridecan-13-yl)amino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₄) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, with a trimethine linkerconnecting the benzopyrylium with the indole group, methyl at R13 andR14, t-butyl at R6, and —COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 3 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 3((E)-2-((2E,4E)-5-(4-tert-butyl-7-(ethyl(2,5,8,11-tetraoxatridecan-13-yl)amino)chromenylium-2-yl)penta-2,4-dienylidene)-1-(5-carboxypentyl)-3,3-dimethylindoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₄) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, with a pentamethine linkerconnecting the benzopyrylium with the indole group, methyl at R13 andR14, t-butyl at R6, and —COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 3 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 13((E)-2-((E)-3-(4-tert-butyl-7-(diethylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methylindoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₁) at position R13, i.e., a methylatedpolyethylene glycol, with a trimethine linker connecting thebenzopyrylium with the indole group, methyl at R14, t-butyl at R6, and—COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 13 (PEG₁), shown below:

In one embodiment, the compound is 682 Compound 13((E)-2-((E)-3-(4-tert-butyl-7-(diethylamino)chromenylium-2-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(2,5,8,11-tetraoxatridecan-13-yl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₄) at position R13, i.e., a methylatedpolyethylene glycol, with a trimethine linker connecting thebenzopyrylium with the indole group, methyl at R14, t-butyl at R6, and—COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 13 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 13((E)-2-((2E,4E)-5-(4-tert-butyl-7-(diethylamino)chromenylium-2-yl)penta-2,4-dienylidene)-1-(5-carboxypentyl)-3-methyl-3-(2,5,8,11-tetraoxatridecan-13-yl)indoline-5-sulfonate),according to general formula I and shown below, which contains apolyethylene glycol (PEG₄) at position R13, i.e., a methylatedpolyethylene glycol, with a pentamethine linker connecting thebenzopyrylium with the indole group, methyl at R14, t-butyl at R6, and—COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 13 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 3((E)-3-(4-(2,5-dioxopyrrolidin-1-yloxy)-4-oxobutyl)-2-((9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)methylene)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a monomethine linker connecting the benzopyrylium with theindole group, and an NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and an NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and an NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3((E)-3-(4-(2,5-dioxopyrrolidin-1-yloxy)-4-oxobutyl)-2-((E)-3-(9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihyldropyrano[3,2-g]quinolin-1-ium-4-yl)allylidene)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a trimethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3((E)-3-(4-(2,5-dioxopyrrolidin-1-yloxy)-4-oxobutyl)-2-((2E,4E)-5-(9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)penta-2,4-dienylidene)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a pentamethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R13:

In embodiments, with an example below, substituents shown innon-limiting examples may be replaced with other disclosed substituents,such as 682 Compound 3, according to general formula II and shown below,which contains a —C(CH₃)₃ (t-butyl) group in place of the above shownphenyl group:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3((E)-3-(4-(2,5-dioxopyrrolidin-1-yloxy)-4-oxobutyl)-2-((2E,4E,6E)-7-(9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)hepta-2,4,6-trienylidene)-3-methyl-1-(3-sulfonatopropyl)indoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a heptamethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 3((Z)-1-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-2-((9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)methylene)-3,3-dimethylindoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a monomethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with amonomethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3((Z)-1-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-2-((E)-3-(9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)allylidene)-3,3-dimethylindoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a trimethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3((Z)-1-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-2-((2E,4E)-5-(9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)penta-2,4-dienylidene)-3,3-dimethylindoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a pentamethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R10:

In various embodiments, and as but one example below, varioussubstituents which have been shown in non-limiting examples may bereplaced with other described substituents, such as 682 Compound 3,according to general formula II and shown below, which contains a—C(CH₃)₃ (t-butyl) group in place of the above shown phenyl group:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3((Z)-1-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-2-((2E,4E,6E)-7-(9-(2-methoxyethyl)-6,8,8-trimethyl-2-phenyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)hepta-2,4,6-trienylidene)-3,3-dimethylindoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) bound to the benzopyrylium by N, at positionR3, i.e., a methylated polyethylene glycol, R1 and R2 form a substitutedring, with a heptamethine linker connecting the benzopyrylium with theindole group, and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₂)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₃)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₄)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₅)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 3, according to generalformula II and shown below, which contains a polyethylene glycol (PEG₆)bound to the benzopyrylium by N, at position R3, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with aheptamethine linker connecting the benzopyrylium with the indole group,and NHS-ester at R10:

In one embodiment, the compound is 682 Compound 13((Z)-2-((2E,4E)-5-(2-tert-butyl-9-ethyl-6,8,8-trimethyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)penta-2,4-dienylidene)-1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methylindoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₁) at position R13, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and —COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 13 (PEG₁), shown below:

In one embodiment, the compound is 682 Compound 13((Z)-2-((2E,4E)-5-(2-tert-butyl-9-ethyl-6,8,8-trimethyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)penta-2,4-dienylidene)-1-(5-carboxypentyl)-3-methyl-3-(2,5,8,11-tetraoxatridecan-13-yl)indoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₄) at position R13, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with apentamethine linker connecting the benzopyrylium with the indole group,and COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 13 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 13((Z)-2-((E)-3-(2-tert-butyl-9-ethyl-6,8,8-trimethyl-8,9-dihydropyrano[3,2-g]quinolin-1-ium-4-yl)allylidene)-1-(5-carboxypentyl)-3-methyl-3-(2,5,8,11-tetraoxatridecan-13-yl)indoline-5-sulfonate),according to general formula II and shown below, which contains apolyethylene glycol (PEG₄) at position R13, i.e., a methylatedpolyethylene glycol, R1 and R2 form a substituted ring, with atrimethine linker connecting the benzopyrylium with the indole group,and

—COOH at R10:

One non-limiting example of an activated compound according to generalformula I is an NHS-ester of 682 Compound 13 (PEG₄), shown below:

In one embodiment, the compound is 682 Compound 1, shown below, whichcontains one ethylene glycol (PEG₁), i.e., a methylated ethylene glycol,bound to the substituted ring formed by R1 and R2, sulfoalkyl at R3 andR10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 1, shown below, whichcontains one ethylene glycol (PEG₂), i.e., a methylated ethylene glycol,bound to the substituted ring formed by R1 and R2, sulfoalkyl at R3 andR10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 1, shown below, whichcontains one ethylene glycol (PEG₃), i.e., a methylated ethylene glycol,bound to the substituted ring formed by R1 and R2, sulfoalkyl at R3 andR10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 1, shown below, whichcontains one ethylene glycol (PEG₄), i.e., a methylated ethylene glycol,bound to the substituted ring formed by R1 and R2, sulfoalkyl at R3 andR10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 1, shown below, whichcontains one ethylene glycol (PEG₅), i.e., a methylated ethylene glycol,bound to the substituted ring formed by R1 and R2, sulfoalkyl at R3 andR10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 1, shown below, whichcontains one ethylene glycol (PEG₆), i.e., a methylated ethylene glycol,bound to the substituted ring formed by R1 and R2, sulfoalkyl at R3 andR10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 2, shown below, whichcontains two ethylene glycols (PEG₁), i.e., a methylated ethyleneglycol, bound to the substituted ring formed by R1 and R2, sulfoalkyl atR3 and R10, and NHS-ester at R13:

In embodiments, with one example below, substituents shown innon-limiting examples may be replaced with other disclosed substituents,such as 682 Compound 2, according to general formula II and shown below,which contains phenyl in place of the above shown —C(CH₃)₃ (t-butyl)group:

In one embodiment, the compound is 682 Compound 2, shown below, whichcontains two ethylene glycols (PEG₂), i.e., a methylated ethyleneglycol, bound to the substituted ring formed by R1 and R2, sulfoalkyl atR3 and R10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 2, shown below, whichcontains two ethylene glycols (PEG₃), i.e., a methylated ethyleneglycol, bound to the substituted ring formed by R1 and R2, sulfoalkyl atR3 and R10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 2, shown below, whichcontains two ethylene glycols (PEG₄), i.e., a methylated ethyleneglycol, bound to the substituted ring formed by R1 and R2, sulfoalkyl atR3 and R10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 2, shown below, whichcontains two ethylene glycols (PEG₅), i.e., a methylated ethyleneglycol, bound to the substituted ring formed by R1 and R2, sulfoalkyl atR3 and R10, and NHS-ester at R13:

In one embodiment, the compound is 682 Compound 2, shown below, whichcontains two ethylene glycols (PEG₆), i.e., a methylated ethyleneglycol, bound to the substituted ring formed by R1 and R2, sulfoalkyl atR3 and R10, and NHS-ester at R13:

In embodiments, with one example below, substituents shown innon-limiting examples may be replaced with other disclosed substituents,such as 682 Compound 2, according to general formula II and shown below,which contains sulfoalkyl in place of the above shown alkyl group:

In embodiments, with one example below, substituents shown innon-limiting examples may be replaced with other disclosed substituents,such as 682 Compound 2, according to general formula II and shown below,which contains a phenyl group in place of the above shown —C(CH₃)₃(t-butyl) group:

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydrophilicity or hydrophobicity. Asulfo group may be added at any site, known to one skilled in the art.Thus, there is no maximum or minimum number of sulfo groups added.

In addition to their ability for direct detection, many fluorescentlabels have the ability to also quench fluorescence of an adjacent,second, fluorescent label, particularly if the second label is held inproximity to the first label by being covalently bound to the samemolecule, such as a protein. In such cases, fluorescence resonanceenergy transfer (FRET) may occur preferential to emission, thusinhibiting fluorescence. Other nonfluorescent analogs of fluorescentmolecules can be designed solely for the purpose of accepting the energytransferred from a neighboring fluorescent label, and such dye analogsare termed quenchers. Quenchers provide a sensitive probe of molecularconformation, binding, and other interactions due to their dependence onthe distance and magnitude of the interaction between the quencher andfluorophore. Quenchers may be used in a variety of assays, described inHermanson (2013) Bioconjugate Techniques, Third Edition, Chapter 1,Section 3.1, Elsevier, 225 Wyman Street, Waltham Mass. In embodiments,quenchers are used in assays labeling a biomolecule, with a fluorophoreand a quencher on opposite ends of the biomolecule. In one embodimentthe biomolecule is a peptide or a protein and the assay measuresprotease activity. In one embodiment fluorescent reporter quencher pairsare used in nucleic acid detection and analysis.

Fluorescent nucleic acid probes are important tools for genetic analysisin both genomic research and development and in clinical medicine. Oneclass of fluorescent probes includes self-quenching probes, also knownas fluorescence energy transfer (FET) probes. The detailed design ofdifferent FET probes may vary, e.g., in one embodiment both afluorophore and a quencher are tethered to nucleic acid with the probeconfigured such that the fluorophore is proximate to the quencher andthe probe produces a signal only as a result of its hybridization to anintended target. One application for probes including areporter-quencher molecule pair is use in nucleic acid amplificationreactions such as polymerase chain reactions (PCR) to detect thepresence and amplification of a target nucleic acid sequence.

The 5′-nuclease PCR assay, also referred to as TaqMan™ assay (Holland etal., Proc. Natl. Acad. Sci. USA 88 (1991) 7276; Lee et al., NucleicAcids Res. 21 (1993) 3761), detects an amplification product withoutprior separation of primer and product. This assay detects accumulationof a specific PCR product by hybridization and cleavage of a doublylabeled fluorogenic “TaqMan” probe during the amplification reaction.The fluorogenic probe is a nucleic acid labeled with both a fluorescentreporter dye and a quencher dye. During PCR, this probe is cleaved bythe 5′-exonuclease activity of DNA polymerase only if it hybridizes tothe segment being amplified. Cleavage of the probe generates an increasein the fluorescence intensity of the reporter dye.

The “beacon probe” method (Tyagi et al. Nature Biotech. 14 (1996) 303;U.S. Pat. No. 5,312,728) is another method to detect amplificationproducts using energy transfer. This method uses nucleic acidhybridization probes that can form hairpin structures. On one end of thehybridization probe, either the 5′- or the 3′-end, is a donorfluorophore. On the other end of the hybridization probe is an acceptormoiety. In this method the acceptor moiety is a quencher, absorbingenergy from the donor. Thus, when the beacon is in the openconformation, the fluorescence of the donor fluorophore is detectable.When the beacon is in the closed hairpin conformation, the fluorescenceof the donor fluorophore is quenched. When used in PCR, the molecularbeacon probe, which hybridizes to one of the strands of the PCR product,is in open conformation and fluorescence is detected, while unhybridizedprobe does not fluoresce. As a result, the amount of fluorescence willincrease as the amount of PCR product increases, and thus is used as ameasure of PCR progress.

Certain limitations impede the application and use of FET probes, orresult in assays that are less sensitive than they could be. Onelimitation is the presence of background fluorescence attributable tothe emission of the quencher, giving the probe a higher than desirablefluorescent noise background. Using a quencher that is not afluorophore, such as derivatives of 4-(dimethylamino)azobenzene (DABCYL)can ameliorate this problem. DABCYL is useful as a quenching agent for alimited group of fluorophores with emission characteristics that overlapthe absorption characteristics of DABCYL. The limited absorption rangeof DABCYL restricts its utility by allowing only a limited number offluorophores in conjunction with DABCYL. Because relatively fewfluorophores can be used with DABCYL in FET pairs, multiplexapplications, where use of two or more fluorophores with clearlyresolved fluorescence emission spectra are desired, are difficult todesign using this quencher.

In view of the limitations of available quenchers and probes, such asFET probes constructed with these quenchers, improved quenchers that canbe incorporated into probes for detecting analytes rapidly, sensitively,reliably, and quantitatively are desired. Ideal quenchers have little tono fluorescent quenching signal. A series of quenchers with similarphysical properties, but distinct spectral properties, would be useful.

In one embodiment, quenchers of excited state energy that aresubstantially non-fluorescent are provided. The inventive compoundsprovide a class of quenchers that are functionalized to allow theirrapid attachment to probe components using techniques known in the artor modifications of such techniques without undue experimentation.

The inventive compounds provide a class of quenchers that can beengineered or tuned to have a desired light absorption profile. Byvarying the number and identity of the substituents of the inventivecompounds, the spectral properties, e.g., absorbance, of a compound canbe “tuned” to match the spectral characteristics, e.g., emission, of oneor more fluorophores.

As used herein, energy transfer refers to the process by which theexcited state energy of an excited group, such as an otherwisefluorescent molecule, is altered by a modifying group, such as toproduce a quenching of the fluorescence. In this manner, if the excitedstate energy-modifying group is a quenching group, the fluorescenceemission from the fluorescent group is attenuated, i.e., quenched.Energy transfer can occur through fluorescence resonance energy transfer(FRET), or through direct energy transfer (FET). The exact energytransfer mechanisms in these two cases are different. Any reference toenergy transfer herein encompasses all of these mechanistically-distinctphenomena.

As used herein, energy transfer pair refers to any two molecules thatparticipate in energy transfer. Typically, one of the molecules acts asa fluorescent group, and the other acts as a fluorescence-modifyinggroup. In one embodiment, the energy transfer pair comprises afluorescent group and a quenching group as described. There is nolimitation on the identity of the individual members of the energytransfer pair; all that is required is that the spectroscopic propertiesof the energy transfer pair as a whole change in some measurable way ifthe distance between the individual members is altered by some criticalamount. Energy transfer pair is used to refer to a group of moleculesthat form a complex within which energy transfer occurs. Such complexesmay include, e.g., two fluorescent groups that may be different from oneanother and one quenching group, two quenching groups and onefluorescent group, or multiple fluorescent groups and multiple quenchinggroups. Where there are multiple fluorescent groups and/or multiplequenching groups, the individual groups may be different from oneanother.

As used herein, fluorescence-modifying group refers to a molecule thatcan alter in any way the fluorescence emission from a fluorescent group.A fluorescence-modifying group generally accomplishes this through anenergy transfer mechanism. Depending on the identity of thefluorescence-modifying group, the fluorescence emission can undergo anumber of alterations, including but not limited to attenuation,complete quenching, enhancement, a shift in wavelength, a shift inpolarity, and a change in fluorescence lifetime. One example of afluorescence-modifying group is a quenching group which, as used herein,refers to any fluorescence-modifying group that can attenuate at leastpartly the light emitted by a fluorescent group. This attenuation isreferred to as quenching. Hence, illumination of the fluorescent groupin the presence of the quenching group leads to an emission signal thatis less intense than expected, or even is completely absent. Quenchingtypically occurs through energy transfer between the fluorescent groupand the quenching group.

In one embodiment, a class of fluorescence modifiers, e.g., quenchers,of excited state energy is provided. These compounds absorb excitedstate energy from a reporter fluorophore, but are themselvessubstantially non-fluorescent. In embodiments, the fluorophoretransferring the excited state energy to the quencher will be a labelthat is attached to an analyte, or a species that interacts with andallows detection and/or quantification of the analyte.

In one embodiment, the quencher contains a benzopyrylium group with aninterrupted aromatic system that uses either a dihydro derivative or anazo bond to prevent fluorescence. In one embodiment, the quencherfurther contains a reactive functional group providing a locus forconjugation of the quencher to a carrier molecule. Although quencherscan be used in their free unbound form, they may be tethered to anotherspecies. Thus, in embodiments, quenchers further comprise a reactivefunctional group that provides a locus for conjugation of the quencherto a carrier molecule.

In embodiments, the disclosed quenchers have substantially no nativefluorescence, particularly near their absorbance maxima or near theabsorbance maxima of fluorophores used in conjunction with thequenchers. In one embodiment, quenchers have an absorbance maximum offrom about 400 nm to about 760 nm. In one embodiment, quenchers have anabsorbance maximum in the ultraviolet (UV) spectral range. In oneembodiment, quenchers have an absorbance maximum in the near infrared(NIR) spectral range.

Selection of appropriate donor-acceptor pairs for particular probes isdisclosed in, e.g., Pesce et al., Eds., Fluorescence Spectroscopy(Marcel Dekker, New York 1971); White et al., Fluorescence Analysis: APractical Approach (Marcel Dekker, New York 1970); etc. Lists offluorescent and chromogenic molecules and their relevant opticalproperties for choosing reporter-quencher pairs are available, e.g.,Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2ndEdition (Academic Press, New York 1971); Griffiths, Colour andConstitution of Organic Molecules (Academic Press, New York 1976);Bishop, Ed., Indicators (Pergamon Press, Oxford 1972); Haugland,Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes,Eugene Oreg. 1992); Pringsheim, Fluorescence and Phosphorescence(Interscience Publishers, New York 1949); etc. Methods for derivatizingreporter and quencher molecules for covalent attachment by commonreactive groups that can be added to a nucleic acid are known, e.g.,Haugland (supra); U.S. Pat. Nos. 3,996,345 and 4,351,760. Thus, oneskilled in the art can select an energy exchange pair for a particularapplication, and can conjugate members of this pair to a probe molecule,e.g., a nucleic acid, peptide, or other polymer, without undueexperimentation.

In one embodiment an absorbance band of the quencher substantiallyoverlaps the fluorescence emission band of the donor. When the donorfluorophore is a component of a probe that uses donor-acceptor energytransfer, the donor fluorescent moiety and the quencher acceptor may beselected so that the donor and acceptor moieties exhibit donor-acceptorenergy transfer when the donor moiety is excited. One factor in choosingthe fluorophore-quencher pair is the efficiency of donor-acceptor energytransfer between them. In one embodiment, the efficiency of FRET betweenthe donor and acceptor moieties is at least 10%, at least 50%, or atleast 80%. The efficiency of FRET can be empirically tested usingmethods known in the art.

The efficiency of energy transfer between the donor-acceptor pair can beadjusted by changing the ability of the donor and acceptor groups todimerize or closely associate. If the donor and acceptor moieties areknown or determined to closely associate, an increase or decrease inassociation can be promoted by adjusting the length of a linker moietyor of the probe itself between the donor and acceptor. The ability ofdonor-acceptor pair to associate can be increased or decreased by tuningthe hydrophobic or ionic interactions, or the steric repulsions in theprobe construct. Thus, intramolecular interactions responsible for theassociation of the donor-acceptor pair can be enhanced or attenuated.Thus, the association between the donor-acceptor pair can be increasedby, e.g., using a donor bearing an overall negative charge and anacceptor with an overall positive charge.

In one embodiment quenchers contain a benzopyrylium group and have aninterrupted aromatic system. Examples of modifications that disrupt thearomatic system of the compound include dihydro derivatives, an azo bond(R—N═N—R′), and addition of one or more phenyl groups at strategiclocations.

In embodiments, as exemplified below, the described compounds can bemodified to disrupt the aromatic system to result in a quencher.

In one embodiment, a pegylated benzopyrylium quencher compound is shownbelow, where R1 is methoxy or a PEG group as described above, R2 issulfoalkyl or a PEG group as described above, and R3 is —OH or —NH—R,where R is a PEG group as described above, where the phenyl and methoxylgroups on the benzopyrylium moiety disrupt the aromatic system.

In one embodiment, a pegylated benzopyrylium quencher compound is shownbelow, where each of R1 and R2 is independently alkyl or a PEG group asdescribed above, R3 is sulfoalkyl or a PEG group as described above, andR4 is —OH or —NH—R, where R is a PEG group as described above, where thephenyl and methyl groups on the benzopyrylium moiety disrupt thearomatic system.

In one embodiment, a pegylated benzopyrylium quencher compound is shownbelow, where each of R1 and R2 is independently alkyl or a PEG group asdescribed above, R3 is sulfoalkyl or a PEG group as described above, andR4 is —OH or —NH—R, where R is a PEG group as described above, where thephenyl and methyl groups on the benzopyrylium moiety disrupt thearomatic system.

The disclosed compounds may be used as dyes for optical labeling, whichmay also be termed marking, of proteins, nucleic acids, oligomers, DNA,RNA, biological cells, lipids, mono-, oligo- and polysaccharides,ligands, receptors, polymers, pharmaceutical or polymer particles andcoupled via functional groups to, for example, HO—, H₂N—, HS—, or HO₂C—function of the substances to be determined, as dyes used in systemsdetermining the quality or quantity of proteins, nucleic acids,oligomers, DNA, RNA, biological cells, lipids, polymers, pharmaceuticalor polymer particles.

This coupling reaction may be in organic or aqueous solutions.

The conjugates from the described compounds and biomolecules displayfluorescing characteristics or deactivate the activated state withoutemitting light (quencher).

The described compounds have use in qualitative and quantitative opticaland in particular optical fluorescent, determinations including immunetests, hybridization procedures, chromatographic or electrophoreticprocedures, FRET systems and high-throughput screening, or for analysisof receptor-ligand change effects on a microarray. General formulas Iand/or II polymethines may be used as dyes for optical marking oforganic or inorganic identification units, e.g., amino acids, peptides,proteins, antigens, haptens, enzyme substrates, enzyme co-factors,biotins, carotinoids, hormones, neuro-hormones, neuro-transmitters,growth factors, lympholocines, lectins, toxins, carbohydrates,oligosaccharides, polysaccharides, dextrans, nucleic acids,oligonucleotides, DNA, RNA, biological cells, lipids, receptor-linkingpharmaceutical or organic or inorganic polymer carriers.

In one embodiment, labeling of probes, also termed identification units,is achieved by forming ionic interactions between general formula Iand/or II complexes and the materials to be labeled.

In one embodiment, the probe or identification unit is linked covalentlywith the described fluorophore. This coupling reaction can be achievedin an aqueous or mainly aqueous solution, preferably at roomtemperature. The resulting probe or conjugate determines the quality orquantity of different biomaterials or other organic and inorganicmaterials through the use of optical procedures.

Both general formula I and/or II complexes and derived systems can beused in qualitative and quantitative optical, and in particular opticalfluorescent, determination procedures for diagnosing cellcharacteristics, e.g., molecular imaging, biosensors, e.g., point ofcare measurements, genome research, and miniaturization technologies.Applications occur in, e.g., cytometry and cell sorting, fluorescencecorrelation spectroscopy (FCS), ultra-high-throughput-screening (UHTS),multicolor fluorescence in situ hybridization (FISH), FRET systems, andmicroarrays e.g., DNA- and protein chips.

A microarray is a grid arrangement of molecules immobilized on at leastone surface used, e.g., to study receptor-ligand change effects. A gridarrangement is more than two different surface molecules immobilized inknown positions in varying pre-defined regions of the surface.

A receptor is a molecule with affinity to a given ligand. Receptors canbe naturally occurring or synthetically produced molecules. They can beused in pure form or in association with another species. They can belinked covalently or non-covalently to a linkage partner either directlyor using certain coupling mediators. Examples of receptors detectableusing the inventive compounds and methods include, but are not limitedto, agonists and antagonists for cell membrane receptors, toxins andother poisonous substances, viral epitopes, hormones such as steroids,hormone receptors, peptides, enzymes, enzyme substrates, activesubstances acting as co-factors, lectins, sugars, oligonucleotides,nucleic acids, oligosaccharides, cells, cell fragments, tissuefragments, proteins and antibodies.

A ligand is a molecule that is recognized by a certain receptor.Examples of ligands that are detectable using the inventive compoundsand methods include, but are not limited to, agonists and antagonistsfor cell membrane receptors, toxins and other poisonous substances,viral epitopes, hormones such as steroids, hormone receptors, peptides,enzymes, enzyme substrates, active substances acting as co-factors,lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,proteins and antibodies.

The disclosed asymmetrical polymethines possess an easily derivatizableheterocycle and a 6 ring heterocycle, i.e., a novel substitution.

Relatively small molecules absorb in the spectral range >550 nm anddisplay fundamentally improved photochemical and thermal stability,compared with previously known polymethines with maximum absorptionmaxima >650 nm (penta- and heptamethines).

It is possible, through molecular engineering, to control the positionand intensity of absorption and emission maxima at will and to adjustthem in line with emission wavelengths of different activating lasers,in particular diode lasers.

The inventive compounds were synthesized by condensing the two differentheterocycles and a C-1, C-3, or C-5 component (“mulligan procedure”).Further manufacturing procedures involve condensation of one of the CHacid heterocycles with the C-1, C-3, or C-5 component at an initialreaction stage and, following isolation of the 1:1 condensation product,conversion to a polymethine with the second CH-acid heterocycle in asubsequent condensation process. The sequence of heterocycleapplications involved is considerable. On this basis, many hydrophilic,variously functionalized dyes differing in total charge and thespecificity/reactivity of the activated groups used for immobilizationcan be easily manufactured in few reaction steps.

Conjugates of the compounds were prepared by covalently coupling thecompounds to a biomolecule using the functional substituent on theN-position of the indole ring. This functional substituent was activatedby routine protein chemistry reaction methods known to one skilled inthe art. The activated compound may be converted to, e.g., and withoutlimitation, N-hydroxysuccinimide (NHS)-ester, acid fluoride,tetrafluorophenyl (TFP)- or sulfotetrafluorophenyl (STP)-ester,iodoacetyl group, maleimide, hydrazide, sulfonyl chloride, orphenylazide. Methods for preparing such compounds are known to oneskilled in the art. In one embodiment, the activated substituent wasthen reacted with an amino group on the biomolecule under conditions toconjugate the desired biomolecule.

In one embodiment, a non-activated carboxyl group on the N-position ofthe indole in the compound was coupled to an amine using a carbodimide.

In one embodiment, a N-hydroxysuccinimidyl ester (X=—NHS) of a compoundwas formed as follows: 20 μmol dye with X=OH (carboxyalkyl group), 8 mg(40 μmol) dicyclohexylcarbodiimide, and 5 mg (40 μmol)N-hydroxysuccinimide were dissolved in 2 ml DMF and 100 μl water. Six μl(40 μmol) triethylamine was added. The reaction mixture was stirred atroom temperature (about 19° C. to about 22° C.) for 24 hours and thenfiltered. The solvent was removed and the residue was washed withdiethylether. The reaction proceeded quantitatively.

In one embodiment, a maleimide (X=—NH—CH₂CH₂-maleimide) of a compound isformed as follows: 20 μmol dye with X=—NHS (N-hydroxysuccinimid-ester)was dissolved in 2 ml DMF and 100 μl water and mixed with 7.6 mg (30μmol) 2-maleimidoethylamine-trifluoracetate and 5 μl (30 μmol)N-ethyldiisopropyl-amine. The reaction mixture is stirred for threehours at room temperature. The solvent was evaporated under reducedpressure. The residue is washed with diethylether and acetone and driedin vacuum. The reaction proceeds quantitatively.

In one embodiment, a iodoacetamide (X=—NH—CH₂CH₂—NH—CO—CH₂—I) of acompound is formed as follows: 20 μmol dye with X=—NHS(N-hydroxysuccinimid-ester) was dissolved in 2 ml DMF and 100 μl water,followed by addition of 40 mg (300 μmol) ethylendiamindihydrochlorideand 26 μl (150 μmol) N-ethyldiisopropyl-amine. The reaction mixture isstirred for three hours at room temperature. The solvent is thenevaporated under reduced pressure, the residue was dissolved inmethanol, and the ethylendiamindihydrochlorid was removed by filtration.Methanol is evaporated under reduced pressure. The residue is dissolvedin 2 ml dry DMF, followed by addition of 7 mg (25 μmol) N-succinimidyliodoacetate and 4 μl (25 μmol) N-ethyldiisopropylamine. The reactionmixture is stirred for three hours at room temperature. The solvent isevaporated under reduced pressure and the residue purified by reversephase HPLC.

In one embodiment, a hydroxyl group, such as a terminal hydroxyl group,can be subsequently activated to a reactive derivative able to linkwith, e.g., proteins and other molecules. Examples of activating groupsinclude tosyl chloride (TsCl), tresyl chloride (TrCl), disuccinimidylcarbonate (DSC), divinyl sulfone, bis-epoxy compounds, carbonyldiimidazole (CDI), 2-fluoro-1-methylpyridinium (FMP), andtrichloro-s-triazine (TsT). In one embodiment, the hydroxyl group isactivated to a succinimidyl carbonate, which is reactive with amines.

Coupling between the compound and the biomolecule may be performed asfollows. The compound was reacted with the biomolecule in an organic oraqueous solution at pH between pH 5-12 inclusive. The compound need notbe dissolved in an organic solvent, such as dimethyl formamide (DMF) ordimethyl sulfoxide (DMSO), prior to adding the biomolecule. In oneembodiment, coupling may be performed in a 100% aqueous solution. In oneembodiment, the coupling reaction occurs at room temperature (about 19°C. to about 22° C.).

To form a composition or dye, at least one biocompatible excipient wasadded to the compound(s), as known to one of ordinary skill in the art.Excipients include, but are not limited to, buffers, solubilityenhancing agents, stabilizing agents, etc.

In one embodiment, a kit for performing an assay method contains atleast one inventive compound, and instructions for performing the methodusing the compound.

The disclosed activated compounds, i.e., the compound modified with areactive group, are useful to label macromolecules, e.g., antibodies,streptavidin, etc., using methods known to one skilled in the art, e.g.,Hermanson, Bioconjugate Techniques, 2nd Ed., London, Elsevier Inc. 2008.The reaction was carried out for one to two hours at room temperature,and then desalted by dialyzing against several changes of phosphatebuffered saline (pH 7.2) or purified by gel filtration to remove theunreacted fluorescent dye. The resulting compound-biomolecule conjugatewas used to detect, e.g., specific proteins in immunoassays, sugars inglycoproteins with lectins, protein-protein interactions,oligonucleotides in nucleic acid, hybridization, and in electrophoreticmobility shift assays (EMSA).

The resulting compound-biomolecule conjugates exhibited fluorescentproperties and could be used in optical methods including fluorescenceoptical qualitative and quantitative determination methods such asmicroscopy, immunoassays, hybridization methods, chromatographic andelectrophoretic methods, fluorescence resonance energy transfer (FRET)systems, high throughput screenings, analysis of receptor-ligandinteractions on a microarray, etc.

Compounds in any embodiment were used as dyes for optical labelling ormarking of organic or inorganic biomolecules, referred to as recognitionunits. Recognition units are molecules having specificity and/oraffinity for a specific group of molecules. Examples include, but arenot limited to, antibodies that have affinity for antigens, enzymes thatbind and/or react with a specific bond or bonds within a sequence ofamino acids in a peptide or react with a substrate, cofactors such asmetals that enhance or inhibit specific interactions, lectins that bindspecific sugars or sugar sequences (e.g., oligosaccharides,polysaccharides, dextrans, etc.), biotin binding proteins such as avidinand streptavidin that bind biotin and biotinylated molecules, antibodybinding proteins such as Protein A, Protein G, Protein A/G and ProteinL, sequences of amino acids or metals that have affinity for each other(e.g., histidine sequences that bind nickel or copper, phosphatecontaining proteins that bind gallium, aluminium, etc.), specificsequences of nucleic acids such as DNA and/or RNA oligonucleotides thathave affinity for proteins, specific sequences of amino acids that haveaffinity for DNA and/or RNA, haptens, carotenoids, hormones (e.g.,neurohormones), neurotransmitters, growth factors, toxins, biologicalcells, lipids, receptor binding drugs or organic or inorganic polymericcarrier materials, fluorescent proteins such as phycobilliproteins(e.g., phycoethrin, allophycocyanin), etc. Ionic interactions betweenrecognition units and the disclosed compounds results in labeling of therecognition units. The recognition unit and compound can be covalentlybound. The result is a conjugate for qualitative or quantitativedetermination of various biomaterials or other organic or inorganicmaterials using optical methods.

The inventive compounds and/or conjugates are used in optical, includingfluorescence optical, qualitative and/or quantitative determinationmethods to diagnose properties of cells, e.g. molecular imaging, inbiosensors, e.g., point of care measurements, for investigation of thegenome, and in miniaturizing technologies. Microscopy, cytometry, cellsorting, fluorescence correlation spectroscopy (FCS), ultra highthroughput screening (UHTS), multicolor fluorescence in situhybridisation (mc-FISH), FRET-systems and microarrays (DNA- andprotein-chips) are exemplary application fields. The inventive compoundshave utility in, e.g., in vivo imaging, ex vivo imaging, multispectraloptoacoustic tomography (MSOT) imaging, photoacoustic imaging andimaging systems, tumor imaging with labeled peptides, NIR fluorescence(NIRF) imaging of labeled silica nanoparticles, NIR in vitro imaging andcharacterization, thermal stability determination, cytotoxicity assays,molecular imaging, UV-VIS-NIR spectroscopy, fluorescence correlationspectroscopy, magnetic resonance imaging (MRI) and applications, DNAsequencing, primer labeling for PCR, two-dimensional (2-D) gelelectrophoresis, flow cytometry/fluorescence-activated cell sorting(FACS), laser scanning confocal microscopy, spectral fluorescenceimaging, fluorescent Western blotting, protein arrays and microarrays,antibody labeling, peptide labeling, single molecule detection,nanoparticle conjugation, and biotin/streptavidin conjugation.

Adding PEG 1-6 at appropriate sites to strategically surround the coredye beneficially affected dye hydrophilicity and performance inbiological applications. Previous attempts to render dyes morehydrophilic and less “sticky” toward biomolecules included the additionof multiple sulfonates and/or relatively longer PEG chains to somelocations on dye molecules. However, the addition of too manysulfonates, while having the effect of increasing the relative watersolubility of dyes, can create undesirable nonspecific binding characterdue to negative charge interactions with positively chargedbiomolecules, particularly proteins. Previous attempts to render dyesmore water soluble by adding longer PEG chains to one or two sites on adye had the detrimental effect of dramatically increasing the molecularweight of the dye, possibly preventing efficient access of dye-labeledantibodies and other dye-labeled targeting molecules to bind with innercellular targets, while also not fully surrounding and masking thehydrophobic dye core structure.

In contrast, the use of such relatively short PEG chain modifications atcritical sites on a dye molecule, limiting the total molecular size oflabeled molecules, resulted in dramatically reduced nonspecificity bymasking the hydrophobic dye core.

General Synthesis of General Formula I, Activated Forms, and SpecificExemplary Compounds

The general procedure for synthesis of compounds according to generalformula I was as follows:

One mmol benzopyrylium salt 1 and 1 mmol indolium compound 2 weredissolved in a mixture of 25 ml acetic acid anhydride and 25 ml aceticacid, then 1 mmol trimethylorthoformate and 1 ml pyridine were added.The solution was stirred for about 30 minutes at about 1400° C. Aftercooling to room temperature, the solvent was removed in vacuum.

The residue was heated to reflux for two hours in a mixture of 10 mlacetone and 10 ml 2 M hydrochloric acid, the reaction solution wasneutralized with NaHCO₃ and the solvent was distilled off in vacuum. Theresidue was purified by column chromatography (reversed phase silica gelRP-18, eluent methanol/water). HPLC chromatography was the finalpurification step.

The general procedure for synthesis of dye-NHS esters was as follows:

Fifty μmol dye, free acid 1 was dissolved in 4 ml of DMF and cooled to0° C. To this solution 60 μmolO-succinimidyl-N,N,N′,N′-tetramethyluronium tetrafluoroborate 2 and 60μmol diisopropylethylamin were added. After 20 minutes at 0° C., thesolvents were distilled off in high vacuum. The residue was purified bycolumn chromatography (RP-18 silica gel; acetonitrile/water).

The general procedure for synthesis of dye-TFP ester was as follows:

Fifty μmol dye, free acid 1 was dissolved in 4 ml DMF and cooled to 0°C. To this solution 0.3 mmol dicyclohexylcarbodiimide 2, 60 μmol2,3,5,6-tetrafluoro-4-hydroxybenzene 3 and then 60 μmoldiisopropylethylamin were added. After ten minutes at 0° C. the reactionmixture was warmed to room temperature and stirred for two hours. Thesolvents were distilled off in high vacuum. The residue was purified bycolumn chromatography (RP-18 silica gel; acetonitrile/water).

The general procedure for synthesis of dye-STP ester was as follows:

Fifty μmol dye, free acid 1 was dissolved in 4 ml DMF and cooled to 0°C. To this solution 60 μmol dicyclohexylcarbodiimide 2, 60 μmol2,3,5,6-tetrafluoro-4-hydroxybenzene sulfonic acid sodium salt 3 andthen 60 μmol diisopropylethylamin were added. After ten minutes at 0°C., the reaction mixture was warmed to room temperature and stirred fortwo hours. The solvents were distilled off in high vacuum. The residuewas purified by column chromatography (RP-18 silica gel;acetonitrile/water).

The general procedure for synthesis of dye-maleimide was as follows:

Fifty μmol dye, free acid 1 was dissolved in 4 ml DMF and cooled to 0°C. To this solution 60 μmol O-succinimidyl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate 2 and 60 μmol diisopropylethylamine were added. After20 minutes at 0° C., 60 μmol N-(2-aminoethyl)-maleimide trifluoroacetatesalt and 60 μmol diisopropylethylamin were added. The reaction mixturewas warmed to room temperature and stirred for two hours. The solventswere distilled off in high vacuum. The residue was purified by columnchromatography (RP-18 silica gel; acetonitrile/water).

The general procedure for synthesis of dye-hydrazide was as follows:

Fifty μmol dye, free acid 1 was dissolved in 4 ml DMF and cooled to 0°C. To this solution 60 μmol O-succinimidyl-N,N,N′,N-tetramethyluroniumtetrafluoroborate 2 and 60 μmol diisopropylethylamin were added. After20 minutes at 0° C., 50 μmol hydrazine monohydrochloride and 60 μmoldiisopropylethylamin were added. The reaction mixture was warmed to roomtemperature and stirred for one hour. The solvents were distilled off inhigh vacuum. The residue was purified by column chromatography (RP-18silica gel; acetonitrile/water).

The following compound (PEG4-682; V08-16072) according to generalformula Ia, was synthesized according to the general procedure forgeneral formula I, PEG4-682:

The following compound according to general formula Ia was synthesizedaccording to general procedure for compound I and general procedure fordye-NHS ester, PEG4-682-NHS-ester:

The following compound according to general formula Ia is synthesizedaccording to general procedure for compound I and general procedure fordye-TFP ester, PEG4-682-TFP-ester:

The following compound according to general formula Ia is synthesizedaccording to general procedure for compound I and general procedure fordye-STP ester, PEG4-682-STP-ester:

The following compound according to general formula Ia is synthesizedaccording to general procedure for compound I and general procedure fordye-maleimide. (PEG4-682-maleimide)

The following compound according to general formula Ia is synthesizedaccording to general procedure for compound I and general procedure fordye-hydrazide, PEG4-682-hydrazide:

The following compound according to general formula Ib is synthesizedaccording to general procedure for compound I, PEG4-682:

The following compound according to general formula Ic was synthesizedaccording to general procedure for compound I, PEG4-682 (V13-06190):

The following compound according to general formula Id is synthesizedaccording to general procedure for compound I, PEG4-682:

The following compound according to general formula Ie is synthesizedaccording to general procedure for compound I, PEG4-682:

The following compound according to general formula If is synthesizedaccording to general procedure for compound I, PEG4-682:

The following compound according to general formula Ig was synthesizedaccording to general procedure for compound I, PEG4-682 (V17-03019):

The following compound according to general formula Ig was synthesizedaccording to general procedure for compound I, (PEG4)₂-681 (V03-07005):

The following compound according to general formula Ih is synthesizedaccording to general procedure for compound I, PEG4-682:

The following compound according to general formula Ig is synthesizedaccording to general procedure for compound I, (PEG4)₂-703:

General Synthesis of General Formula II, Activated Forms, and SpecificExemplary Compounds

The general procedure for synthesis of dyes according general formula IIwas as follows:

One mmol benzopyrylium salt 1 and 1 mmol indolium compound 2 weredissolved in a mixture of 25 ml acetic acid anhydride and 25 ml aceticacid, then 1 mmol trimethylorthoformate and 1 ml pyridine were added.The solution was stirred for about 30 min at about 140° C. After coolingto room temperature, the solvent was removed in vacuum.

The residue was heated to reflux for two hours in a mixture of 10 mlacetone and 10 ml 2 M hydrochloric acid, the reaction solution wasneutralized with NaHCO₃ and the solvent was distilled off in vacuum. Theresidue was purified by column chromatography (reversed phase silica gelRP-18, eluent methanol/water). HPLC chromatography was used as finalpurification step.

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II and general procedure fordye-NHS ester, PEG4-632-NHS ester:

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II and general procedure fordye-TFP ester, PEG4-632-TFP ester:

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II and general procedure fordye-STP ester, PEG4-632-STP ester:

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II and general procedure fordye-maleimide, PEG4-632-maleimide

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II and general procedure fordye-hydrazide, PEG4-632-hydrazide:

The following compound according to general formula IIb is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIc is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IId is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIe is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIf is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIg is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIg is synthesizedaccording to general procedure for compound II, (PEG4)₂-631:

The following compound according to general formula IIh is synthesizedaccording to general procedure for compound II, PEG4-632:

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II, PEG4-650:

The following compound according to general formula IIa is synthesizedaccording to general procedure for compound II, PEG4-675:

The following compound according to general formula IIc is synthesizedaccording to general procedure for compound II, PEG4-635:

The following non-limiting examples further describe the compounds,methods, compositions, uses, and embodiments. Vacuum is 30-150 mbarpressure range. Liquid mixing ratios are by volume. NHS isN-hydroxy-succinimide. DCC is dicyclohexylcarbodiimide. DMF isN,N-dimethylformamide.

SYNTHESIS EXAMPLE 1 Synthesis of DY-631

180 mg (0.5 mmol)2-tert-butyl-7-diethylamino-4-methyl-chromenylium-tetrafluoro-borate and242 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were dissolved in 50 ml acetanhydride, with 75 μl (0.6 mmol)trimethylorthoformate and 1 ml pyridine. The solution was stirred forabout 30 min at about 140° C. After cooling to room temperature, thesolvent was removed in the vacuum.

The residue was heated to reflux for two hours in a mixture of 10 mlacetone and 10 ml of 2 M hydrochloric acid, the reaction solutionneutralized with NaHCO₃ and the solvent distilled in the vacuum. Theresidue was chromatographed (SiO₂ RP-18, eluent methanol/water 6:4).

145 mg (39%) yield-UV/Vis (ethanol) λ_(max) (ε)=637 nm (185.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=658 nm.-MS (ESI⁻): 713.2 [M]⁻; 356.4[M−H]²⁻. —C₃₆H₄₅N₂O₉S₂Na (736.88).

SYNTHESIS EXAMPLE 2 Synthesis of DY-631 N-Hydroxysuccinimidyl Ester

15 mg DY-631, 14 mg DCC, 4 mg NHS and 10 μl pyridine were dissolved in 2ml DMF and stirred at room temperature for 24 h. The solvent was removedin vacuum. The residue was washed with diehtylether and dried in vacuum.The reaction was quantitative.

SYNTHESIS EXAMPLE 3 Synthesis of DY-636

206 mg (0.5 mmol)10-tert-butyl-8-methyl-2,3,5,6-tetrahydro-1H,4H-11-oxonia-3a-aza-benzo[de]anthracene-tetrafluoroborateand 242 mg (0.5 mmol)3-(3-ethoxy-carbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accord with example 1.

135 mg (36%) yield-UV/Vis (ethanol) λ_(max) (ε)=645 nm (155.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=670 nm.-MS (ESI⁻): 737.1 [M]⁻; 368.4[M−H]²⁻. —C₃₈H₄₅N₂O₉S₂Na (760.91).

EXAMPLE 4 Synthesis of DY-636 N-Hydroxysuccinimidyl Ester

15 mg DY-636, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

SYNTHESIS EXAMPLE 5 Synthesis of DY-651

206 mg (0.5 mmol)2-tert-butyl-8-ethyl-4,5,7,7-tetramethyl-7,8-dihydro-1-oxonia-8-aza-anthracene-tetrafluoroborateand 242 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accordance with example 1.

145 mg (38%) yield-UV/Vis (Ethanol) λ_(max) (ε)=653 nm (160.000 l·mol⁻¹cm⁻¹).-fluorescence λ_(em)=678 nm.-MS (ESI⁻): 765.1 [M]⁻; 382.4 [M−H]².—C₄₀H₄₉N₂O₉S_(Na) (888.96).

SYNTHESIS EXAMPLE 6 Synthesis of DY-651 N-Hydroxysuccinimidyl Ester

15 mg DY-651, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

SYNTHESIS EXAMPLE 7 Synthesis of DYQ-661

196 mg (0.5 mmol)7-diethylamino-3,4-dimethyl-2-phenyl-chromenylium-tetra-fluoroborate and242 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accord with example 1.

145 mg (37%) yield-UV/Vis (Ethanol) λ_(max) (ε)=661 nm (116.000 l·mol⁻¹cm⁻¹).-MS (ESI⁻): 747.1 [M]⁻, 373.6 [M−H]²⁻. —C₃₉H₄₃N₂O₉S₂Na (770.90).

SYNTHESIS EXAMPLE 8 Synthesis of DYQ-661 N-Hydroxysuccinimidyl Ester

15 mg DYQ-661, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

SYNTHESIS EXAMPLE 9 Synthesis of DY-676

216 mg (0.5 mmol)8-ethyl-4,5,7,7-tetramethyl-2-phenyl-7,8-dihydro-1-oxonia-8-aza-anthracene-tetrafluoroborateand 242 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accord with example 1.

150 mg (37%) yield-UV/Vis (Ethanol) λ_(max) (ε)=674 nm (84.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=699 nm.-MS (ESI⁺): 785.5 [M]⁺.—C₄₂H₄₅N₂O₉S₂Na (807.95).

SYNTHESIS EXAMPLE 10 Synthesis of DY-676 N-Hydroxysuccinimidyl Ester

15 mg DY-676, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

SYNTHESIS EXAMPLE 11 Synthesis of DY-731

180 mg (0.5 mmol) 2-tertbutyl-7-diethylamino-4-methyl-chromenylium-tetrafluoro-borate und 307 mg(0.5 mmol)3-(3-ethoxycarbonylpropyl)-3-methyl-2-(4-phenyl-aminobuta-1,3-dienyl)-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were dissolved in 50 ml of acetanhydride with 1 ml ofpyridine. The solution was stirred for approx. 30 min at approx. 140° C.After cooling to RT, the solvent was removed in the vacuum.

The residue was refluxed for 2 hours in a mixture of 10 ml acetone and10 ml 2-M hydrochloric acid, the reaction solution neutralized withNaHCO₃ and the solvent distilled in the vacuum. The residue waschromatographed (SiO₂—RP-18, eluent methanol/water—6:4).

120 mg (31%) yield-UV/Vis (ethanol) λ_(max) (ε)=736 nm (225.000l·mol⁻¹·cm¹).-fluorescence λ_(em)=759 nm.-MS (ESI⁻): 739.2 [M]⁻, 369.5[M−H]²⁻. —C₃₈H₄₇N₂O₉S₂Na (762.92).

SYNTHESIS EXAMPLE 12 Synthesis of DY-731 N-Hydroxysuccinimidyl Ester

15 mg DY-731, 14 mg DCC and 4 mg NHS were reacted and processed inaccordance with example 2.

SYNTHESIS EXAMPLE 13 Synthesis of DY-751

206 mg (0.5 mmol)2-tert-butyl-8-ethyl-4,5,7,7-tetramethyl-7,8-dihydro-1-oxonia-8-aza-anthracene-tetrafluoroborateand 307 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-3-methyl-2-(4-phenyl-aminobuta-1,3-dienyl)-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accordance with example 11.

120 mg (29%) yield-UV/Vis (ethanol) λ_(max) (ε)=751 nm (220.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=779 nm.-MS (ESI⁺): 793.1 [M+H]⁺,419.4 [M+2 Na]²⁺, 408.4 [M+H+Na]²⁺, 397.4 [M+2H]²⁺. —C₄₂H₅₁N₂O₉S₂Na(814.99).

SYNTHESIS EXAMPLE 14 Synthesis of DY-751 N-Hydroxysuccinimidyl Ester

15 mg DY-751, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

SYNTHESIS EXAMPLE 15 Synthesis of DY-776

216 mg (0.5 mmol)8-ethyl-4,5,7,7-tetramethyl-2-phenyl-7,8-dihydro-1-oxonia-8-aza-anthracene-tetrafluoroborateand 307 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-3-methyl-2-(4-phenylamino-buta-1,3-dienyl)-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accordance with example 11.

110 mg (26%) yield-UV/Vis (Ethanol) λ_(max) (ε)=771 nm (147.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=801 nm.-MS (ESI⁺): 813.1 [M+H]⁺,429.2 [M+2 Na]²⁺, 418.3 [M+H+Na]²⁺, 407.3 [M+2H]²⁺. —C₄₄H₄₇N₂O₉S₂Na(834.98).

SYNTHESIS EXAMPLE 16 Synthesis of DY-776 N-Hydroxysuccinimidyl Ester

15 mg DY-776, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

Examples 17-22 illustrate a general synthesis scheme for formula Icompounds.

EXAMPLE 17 Synthesis of DY-681

180 mg (0.5 mmol)4-tert-butyl-7-diethylamino-2-methyl-chromenylium-tetrafluoro-borate and242 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accordance with example 1.

140 mg (39%) yield-UV/Vis (Ethanol) λ_(max) (ε)=691 nm (125.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=708 nm.-MS (ESI⁻): 713.2 [M]⁻; 356.4[M−H]²⁻. —C₃₆H₄₅N₂O₉S₂Na (736.88).

SYNTHESIS EXAMPLE 18 Synthesis of DY-681 N-Hydroxysuccinimidyl Ester

15 mg DY-681, 14 mg DCC and 4 mg NHS were reacted and processed inaccordance with example 2.

SYNTHESIS EXAMPLE 19 Synthesis of DY-701

196 mg (0.5 mmol)7-diethylamino-2,3-dimethyl-4-phenyl-chromenylium-tetrafluoro-borate and242 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-2,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were reacted and processed in accordance with example 1.

150 mg (39%) yield-UV/Vis (Ethanol) λ_(max) (ε)=706 nm (115.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=731 nm.-MS (ESI⁻): 747.2 [M]⁻; 373.4[M−H]²⁻. —C₃₉H₄₃N₂O₉S₂Na (770.90).

SYNTHESIS EXAMPLE 20 Synthesis of DY-701 N-Hydroxysuccinimidyl Ester

15 mg DY-701, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

SYNTHESIS EXAMPLE 21 Synthesis of DY-781

180 mg (0.5 mmol)4-tert-butyl-7-diethylamino-2-methyl-chromenylium-tetrafluoro-borate and307 mg (0.5 mmol)3-(3-ethoxycarbonylpropyl)-3-methyl-2-(4-phenyl-aminobuta-1,3-dicnyl)-5-sulfonato-1-(3-sulfonatopropyl)-3H-indoliumsodium salt were made to react and processed in accordance with example11.

125 mg (33%) yield-UV/Vis (Ethanol) λ_(max) (ε)=783 nm (98.000l·mol⁻¹·cm⁻¹).-fluorescence λ_(em)=800 nm.-MS (ESI⁺): 785.3 [M+Na]⁺;763.3 [M+H]⁺; 404.4 [M+2Na]²⁺; 393.5 [M+H+Na]²⁺. —C₃₈H₄₇N₂O₉S₂Na(762.92).

SYNTHESIS EXAMPLE 22 Synthesis of DY-781 N-Hydroxysuccinimidyl Ester

15 mg DY-781, 14 mg DCC and 4 mg NHS were reacted and processed inaccord with example 2.

EXAMPLE 1

The following properties of V03-07005, V08-16072, V13-06190, andV17-03019 were compared with commercially available dyes. DyLight 680NHS is a non-pegylated benzopyrylium dye and the ‘V’ dyes are pegylateddyes described above.

Alexa DyLight V03-07005- V08-16072- V13-06190- V17-03019- Fluor 680-NHSNHS NHS NHS NHS 680-NHS MW (g/mol) 950 1158.33 1140.25 1274.38 1112.22~1150 Ex (nm) 682 690 691 691 691 679 (EtOH) (EtOH) (EtOH) (EtOH) 675679 679 675 (PBS)* (PBS)* (PBS)* (PBS)* Em (nm) 715 708 712 712 712 702ε 140,000 140,000 140,000 140,000 140,000 184,000 (M⁻¹cm⁻¹)(theoretical) PEG 0 4/2 4/1 4/2 4/1 N/A (length/# of (on same N)(different N) chain) Sulfonate 3 2 3 3 3 3 *Excitation of V03-07005,V08-16072, V13-06190, and V1703019 NHS was shifted from 690 nm inethanol to about 675 nm in PBS.

EXAMPLE 2

Inventive and commercial compounds, each as the NHS ester, wereconjugated to goat anti-mouse (GAM) and goat anti-rabbit (GAR)antibodies. GAM and GAR, at 10 mg/ml in phosphate buffered saline (PBS),were dialyzed against 50 mM borate buffer, pH 8.5. The compounds werereconstituted in dimethylformamide (DMF) at 10 mg/ml and combined at2.5×, 5×, 7.5×, 10×, and 15× molar excess with GAM or GAR for 65 minutesat room temperature to label the antibodies.

The labeled compounds, also termed dyes or labels, were subjected toPierce Dye Removal Resin (PDDR) to remove the unlabeled (free) compound;100 μl of the packed resin was used per mg protein purified. Thepurified antibody-labeled dyes were then diluted by adding 150 μl PBS.All conjugates were diluted 1:20 and scanned for absorbance from 700 nmto 230 nm to determine the protein concentration, and to determine themole dye to mole protein ratio on a UV Cary spectrophotometer. Labelingefficiency, indicated as dye to protein ratio (D/P), was compared, withresults showing degree of labeling below.

2.5X 5X 10X 15X GAM-V03-07005 1.5 2.6 4.0 3.9 GAR-V03-07005 1.3 2.5 4.23.7 GAM-V08-16072 1.4 2.5 4.1 5.8 GAR-V08-16072 1.6 2.5 3.9 4.8GAM-V13-06190 1.5 2.7 4.0 5.2 GAR-V13-06190 1.2 2.5 4.1 5.1GAM-V17-03019 1.3 2.2 3.9 4.7 GAR-V17-03019 1.1 2.2 3.1 3.2 GAM-DyLight680 1.3 2.5 2.7 2.1 GAR-DyLight 680 1.2 2.0 2.6 2.1 GAM-Alexa Fluor-6801.6 2.2 4.4 7.0 GAR-Alexa Fluor-680 1.7 2.9 4.7 4.8 D/P GAM-AlexaFluor-680 5.0 GAM-DyLight 680 3.1Labeling efficiency of GAM and GAR was similar for all benzopyryliumdyes at low molar excesses.

EXAMPLE 3

Performance of dye-GAM and dye-GAR conjugates was evaluated in afunctional assay. Wells of a 96-well black clear-bottom plate werecoated with target protein mouse or rabbit IgG immunoglobulin. Onehundred μl mouse or rabbit IgG, at 10 μg/ml, was applied to thecorresponding wells in row 1. The target proteins were serially diluted1:1 from the wells in rows 2 to 7 using 100 μl PBS. One hundred μl ofsamples from the wells in row 7 were discarded. One hundred μl PBS wasadded to the wells in row 8. Plates were incubated overnight at 4° C.and then blocked 2×200 μl with Thermo Scientific SuperBlock® BlockingBuffer. The coated plates were washed 2×200 μl with PBS-Tween and 1×200μl with PBS. Based on the calculated concentrations, conjugates werediluted 1:250 (of 1 mg/ml) in PBS, added to the corresponding plates(100 μl/well) and then incubated for one hour in the dark. The plateswere washed with 2×200 μl with PBS-Tween and 1×200 μl with PBS andfilled with PBS buffer (100 μl/well) prior to scanning the blackclear-bottom plates on LiCor Odyssey at 700 channel, to detectfluorescence intensity.

As shown in FIGS. 1-10, RFU and/or signal to background ratio (S/B) ofthe dyes conjugated to the indicated antibody were compared at variousconcentrations, using the indicated conjugation conditions.

FIG. 1 shows binding intensity results expressed as RFU of a functionalassay using GAR conjugated with 5× molar excess of V03-07005 (greendiamond; plate column 1 and 2), V08-16072 (blue triangle; plate column 3and 4), V13-06190 (purple diamond; plate column 5 and 6), V17-03019(orange circle; plate column 7 and 8), DyLight 680 (red square; platecolumn 9 and 10), and Alexa Fluor 680 (black square; plate column 11 and12). FIG. 2 shows an image of the plate of FIG. 1. Based on the data, at5× molar excess, Alexa Fluor 680-GAR showed the highest bindingintensity. All pegylated benzopyrylium-GAR conjugates performed betterthan non-pegylated DyLight 680-GAR conjugates.

FIG. 3 shows binding intensity results expressed as RFU of a functionalassay using GAR conjugated with 10× molar excess of V03-07005 (greendiamond; plate column 1 and 2), V08-16072 (blue triangle; plate column 3and 4), V13-06190 (purple diamond; plate column 5 and 6), V17-03019(orange circle; plate column 7 and 8), DyLight 680 (red square; platecolumn 9 and 10), and Alexa Fluor 680 (black square; plate column 11 and12). FIG. 4 shows an image of the plate of FIG. 3. Based on the data, at10× molar excess, V03-07005-GAR showed the highest binding intensity.Non-pegylated DyLight 680-GAM showed the lowest binding intensity.

FIG. 5 shows LiCOR Odyssey results expressed as S/B of a functionalassay using GAR conjugated with 5× molar excess (blue bars) or 10× molarexcess (yellow bars) of the indicated compound.

FIG. 6 shows binding intensity results expressed as RFU of a functionalassay using GAM conjugated with 5× molar excess of V03-07005 (greendiamond; plate column 1 and 2), V08-16072 (blue triangle; plate column 3and 4), V13-06190 (magenta diamond; plate column 5 and 6), V17-03019(orange circle; plate column 7 and 8), DyLight 680 (red square; platecolumn 9 and 10), and Alexa Fluor 680 (black square; plate column 11 and12). FIG. 7 shows an image of the plate of FIG. 6. Based on the data, at5× molar excess, Alexa Fluor 680-GAM showed the highest bindingintensity. V03-07005 performed best of the benzopyrylium dyes.

FIG. 8 shows binding intensity results expressed as RFU of a functionalassay using GAM conjugated with 10× molar excess of V03-07005 (greendiamond; plate column 1 and 2), V08-16072 (blue triangle; plate column 3and 4), V13-06190 (purple diamond; plate column 5 and 6), V17-03019(orange circle; plate column 7 and 8), DyLight 680 (red square; platecolumn 9 and 10), and Alexa Fluor 680 (black square; plate column 11 and12). FIG. 9 shows an image of the plate of FIG. 8. Based on the data, at10× molar excess, GAM conjugated to V03-07005 and V17-03019 dyes showedsimilar binding intensity as Alexa Fluor 680-GAM. These dyes performedbetter than V08-16072 and V013-06190. Non-pegylated DyLight 680-GAMshowed the lowest binding intensity.

FIG. 10 shows LiCOR Odyssey results expressed as S/B of a functionalassay using GAM conjugated with 5× molar excess (blue bars) or 10× molarexcess (beige bars) of the indicated compound.

EXAMPLE 4

In the indicated experiments, the inventive compounds and commercial dyewere evaluated for immunofluorescence in a cell based assay using thefollowing protocol. Frozen U20S cell plates stored at −80° C. werethawed for 30 minutes at 50° C. Storage buffer (PBS) was removed and thecells were permeabilized for 15 minutes with 0.1% Triton-X100 in 1×PBSbuffer (100 μl/well). The cell plate was blocked for 60 minutes in 2%BSA/PBS-0.1% Triton-X100. Primary antibody, either rabbit anti-lamin B1or rabbit anti-HDAC2 (10 μg/ml), diluted in 2% BSA/PBS-0.1% Triton-X100was added to the plate and incubated for one hour at room temperature.Control wells contained only 2% BSA/PBS-0.1% Triton-X100 blocker. Afterincubation, antibody solution was removed from the plate and the platewas washed three times with 100 μl/well PBS-0.5% Tween-20 and one timewith 100 μl/well PBS. GAR secondary antibodies labeled with variousmolar excess of the inventive or commercial compound were diluted to 4μg/ml in PBS and incubated for one hour at room temperature. Plates werewashed three times with 100 μl/well PBST and once with 100 μl/well PBS,and Hoechst (diluted to 0.1 μg/ml in PBS) was added to each well (100μl/well). The plates were scanned on ArrayScan Plate Reader.

FIG. 11 shows detection of lamin B1 in U20S cells with V03-07005-GAR(column 1) and V08-16072-GAR (column 3) at a 2.5× molar excess (row A),5× molar excess (row B), 10× molar excess (row C), and 15× molar excess(row D); and associated controls (columns 2 and 4). FIG. 12 showsdetection of lamin B1 in U20S cells with V13-06190-GAR (column 1) andV17-03019-GAR (column 3) at a 2.5× molar excess (row A), 5× molar excess(row B), 10× molar excess (row C), and 15× molar excess (row D); andassociated controls (columns 2 and 4). FIG. 13 shows detection of laminB1 in U20S cells with DyLight 680-GAR (column 1) and Alexa Fluor 680-GAR(column 3) at a 2.5× molar excess (row A), 5× molar excess (row B), 10×molar excess (row C), and 15× molar excess (row D); and associatedcontrols (columns 2 and 4). Overall, benzopyrylium dye conjugates showedbrighter staining than DyLight 680, and all conjugates performed betterthan Alexa-Fluor 680 conjugate at similar D/Ps. Benzopyrylium dyesperformed best at low molar excesses (2.5-5×). The best performingbenzopyrylium dye was V03-07005, which has two PEG₄ on the samenitrogen. Non-specific binding in the absence of primary antibody isobserved only with Alexa Fluor 680, and is increased with the amount ofdye used.

Quantitative analysis of the data of FIGS. 11-13, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a nucleus, isshown below; “Neg” indicates negative control.

V03-07005- V08-16072- V13-06190- V17-03019- DyLight Alexa Fluor- GAR GARGAR GAR 680-GAR 680-GAR Lamin Lamin Lamin Lamin Lamin Lamin B1 Neg B1Neg B1 Neg B1 Neg B1 Neg B1 Neg  2.5X 236323 3186 209340 2933 1451332993 143740 3177 131853 3256 27862 3004  5X 271178 3093 158162 3146153454 3041 184109 2809 112778 3331 37292 3135 10X 235611 3211 1203703075 103925 2926 152450 3649 66049 2797 64073 5907 15 X 154466 3217106816 3275 98440 3217 103931 2412 73837 3752 66025 9958

Quantitative analysis of the data of FIGS. 11-13, expressed as S/B, isshown below.

V03- V08- V13- V17- Alexa 07005- 16072- 06190- 03019- DyLight Fluor- S/BGAR GAR GAR GAR 680-GAR 680-GAR  2.5 X 74.2 71.4 48.5 45.2 40.5 9.3  5 X87.7 50.3 50.5 65.5 33.9 11.9 10 X 73.4 39.1 35.5 41.8 23.6 10.8 15 X48.0 32/6 30.6 43.1 19.7 6.6

Based on S/B data, the best performing benzopyrylium dye was V03-07005,with two PEG₄ and two sulfo groups, followed by V17-03019. Allbenzopyrylium dyes quenched at high molar excesses, with V08-16072 andV13-06190 gradually quenching from 2.5× molar excess condition. AlexaFluor 680 began quenching at molar excesses greater than 10×. Due tostrong non-specific binding at higher molar excesses, Alexa-680 showeddecreasing signal to background.

FIG. 14 shows nuclear detection of HDAC2 in U20S cells withV03-07005-GAR (column 1) and V08-16072-GAR (column 3) at a 2.5× molarexcess (row A), 5× molar excess (row B), 10× molar excess (row C), and15× molar excess (row D); and associated controls (columns 2 and 4).FIG. 15 shows nuclear detection of HDAC2 in U20S cells withV13-06190-GAR (column 1) and V17-03019-GAR (column 3) at a 2.5× molarexcess (row A), 5× molar excess (row B), 10× molar excess (row C), and15× molar excess (row D); and associated controls (columns 2 and 4).FIG. 16 shows nuclear detection of HDAC2 in U20S cells with DyLight680-GAR (column 1) and Alexa Fluor 680-GAR (column 3) at a 2.5× molarexcess (row A), 5× molar excess (row B), 10× molar excess (row C), and15× molar excess (row D); and associated controls (columns 2 and 4).Conjugates made with pegylated benzopyrylium dyes showed brighterstaining than conjugates with DyLight 680, and all conjugates performedbetter than Alexa-Fluor 680 conjugate at similar D/Ps. Benzopyryliumdyes performed best at low molar excesses. The best performingbenzopyrylium dye was V03-07005, with two PEG₄ on the same nitrogen.Non-specific binding in the absence of primary antibody is observed onlywith Alexa Fluor 680, where background increased with the amount of dyeused.

Quantitative analysis of the data of FIGS. 14-16, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a nucleus, isshown below; “Neg” indicates negative control.

V03-07005- V08-16072- V13-06190- V17-03019- DyLight Alexa Fluor- GAR GARGAR GAR 680-GAR 680-GAR HDAC2 Neg HDAC2 Neg HDAC2 Neg HDAC2 Neg HDAC2Neg HDAC2 Neg  2.5X 80067 2158 86477 2150 79222 2142 83377 2043 644372116 22746 2167  5X 104865 2127 72981 2136 73258 2192 101760 1978 543362194 43923 2512 10X 88650 2488 44540 2111 43725 2337 68080 2303 312951817 65089 2839 15X 47052 2213 28685 2327 36184 2344 38914 1749 200772466 44209 6320

Quantitative analysis of the data of FIGS. 14-16, expressed as S/B, isshown below.

V03- V08- V13- V17- Alexa 07005- 16072- 06190- 03019- DyLight Fluor- S/BGAR GAR GAR GAR 680-GAR 680-GAR  2.5X 37.1 40.2 37.0 40.8 30.4 10.5  5X49.3 34.2 33.4 51.5 24.8 17.5 10X 35.6 21.1 18.7 29.6 17.2 22.9 15X 21.312.3 15.4 22.3 8.1 7.0

The best performing benzopyrylium dye was V03-07005, with two PEG₄ andtwo sulfo groups, then, V17-03019. All benzopyrylium dyes quenched athigh molar excesses, with V08-16072 and V13-06190 gradually quenchingfrom 2.5× molar excess condition. Alexa Fluor 680 began quenching atmolar excesses greater than 10×. Due to the strong non-specific bindingat higher molar excesses, Alexa-680 showed decreasing signal tobackground.

In the indicated experiments, the inventive compounds and commercial dyewere evaluated for immunofluorescence in a cell based assay using thefollowing protocol. Frozen U20S cell plates which were stored at −80° C.were thawed for 30 minutes at 50° C. Storage buffer (PBS) was removedand cells were permeabilized for 15 minutes with 0.1% Triton-X100 in1×PBS buffer (100 μl/well). The cell plate was blocked for 60 minutes in2% BSA/PBS-0.1% Triton-X100. Primary antibody, either mouse anti-ezrin(1.3 μg/ml) or mouse anti-NCoR2 (20 μg/ml), diluted in 2% BSA/PBS-0.1%Triton-X100 was added to the plate and incubated for one hour at roomtemperature. Control wells contained only 2% BSA/PBS-0.1% Triton-X100blocker. After incubation, the antibody solution was removed from theplate and the plate was washed three times with 100 μl/well of PBS-0.5%Tween-20 and one time with 100 μl/well PBS. GAM secondary antibodieslabeled with various molar excess of the inventive or commercialcompound were diluted to 4 μg/ml in PBS and incubated for one hour atroom temperature. The plates were washed three times with 100 μl/well ofPBST and once with 100 μl/well PBS, and Hoechst (diluted to 0.1 μg/ml inPBS) was added to each well (100 μl/well). The plates were scanned onArrayScan Plate Reader.

FIG. 17 shows detection of ezrin in U20S cells with V03-07005-GAM(column 1) and V08-16072-GAM (column 3) at a 2.5× molar excess (row A),5× molar excess (row B), 10× molar excess (row C), and 15× molar excess(row D); and associated controls (columns 2 and 4). FIG. 18 showsdetection of ezrin in U20S cells with V13-06190-GAM (column 1) andV17-03019-GAM (column 3) at a 2.5× molar excess (row A), 5× molar excess(row B), 10× molar excess (row C), and 15× molar excess (row D); andassociated controls (columns 2 and 4). FIG. 19 shows detection of ezrinin U20S cells with DyLight 680-GAM (column 1) and Alexa Fluor 680-GAM(column 3) at a 2.5× molar excess (row A), 5× molar excess (row B), 10×molar excess (row C), and 15× molar excess (row D); and associatedcontrols (columns 2 and 4). The conjugates made with the benzopyryliumdyes showed brighter staining than DyLight 680, and all conjugatesperformed better than Alexa-Fluor 680 conjugate at similar D/Ps. Thebenzopyrylium dyes performed best at low molar excesses. The bestperforming benzopyrylium dye was V03-07005. Non-specific binding in theabsence of primary antibody was observed only with Alexa Fluor 680, andincreased with the amount of dye used.

Quantitative analysis of the data of FIGS. 17-19, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a

V03- V08-16072- V13-06190- V17- DyLight Alexa Fluor- 07005-GAM GAM GAM03019-GAM 680-GAM 680-GAM Ezrin Neg Ezrin Neg Ezrin Neg Ezrin Neg EzrinNeg Ezrin Neg  2.5X 92921 18678 69367 16412 81937 16244 85736 1803961819 16866 44076 20621  5X 117420 17542 79695 16082 89901 15736 8840416994 66550 19346 115695 21620 10X 100740 16998 78398 16434 89417 17205117698 17612 62670 18444 212611 42689 15X 91676 16926 67010 17775 5943622790 85383 18930 39345 24576 204288 74581

Quantitative analysis of the data of FIGS. 17-19, expressed as S/B, isshown below.

V03- V08- V13- V17- Alexa 07005- 16072- 06190- 03019- DyLight Fluor- S/BGAR GAR GAR GAR 680-GAR 680-GAR  2.5X 5.0 4.2 5.0 4.8 3.7 2.1  5X 6.75.0 5.7 5.2 3.4 5.4 10X 5.9 4.8 5.2 6.7 3.4 5.0 15X 5.4 3.8 2.6 4.5 1.62.7

The best performing benzopyrylium dye was V03-07005, with two PEG4 andtwo sulfo groups, then, V17-03019. All benzopyrylium dyes quenched athigh molar excesses. Alexa Fluor 680 began quenching at molar excessesgreater than 10×. Due to strong non-specific binding at higher molarexcesses, Alexa-680-GAM showed decreased signal to background.

NCoR2 was detected in U20S cells with V03-07005-GAM, V08-16072-GAM,V13-06190-GAM, V17-03019-GAM, DyLight 680-GAM, and Alexa Fluor 680-GAMat a 2.5×, 5×, 10×, and 15× molar excesses. Quantitative analysis of thedata, expressed as Mean Total Intensity, which is the average totalintensity of all pixels within a defined area or defined primary objectsuch as a nucleus, is shown below, where “Neg” indicates the negativecontrol condition.

V03-07005- V08- V13-06190- V17-03019- DyLight Alexa Fluor- GAM 16072-GAMGAM GAM 680-GAM 680-GAM NCoR2 Neg NCoR2 Neg NCoR2 Neg NCoR2 Neg NCoR2Neg NCoR2 Neg  2.5X 89539 17648 76027 17163 87251 23790 82175 1710476596 30478 81523 22614  5X 107778 17676 70711 18149 104629 18115 8059417864 71774 17394 98164 23026 10X 77837 18395 59411 17553 66955 1747987665 20020 45648 18873 174913 63422 15X 72466 18356 48666 18072 5536316732 80476 17863 46945 24948 190341 85250

Quantitative analysis of the NCoR2 data, expressed as S/B, is shownbelow.

V03- V08- V13- V17- Alexa 07005- 16072- 06190- 03019- DyLight Fluor- S/BGAR GAR GAR GAR 680-GAR 680-GAR  2.5X 5.1 4.4 3.7 4.8 2.5 3.6  5X 6.13.9 5.8 4.5 4.1 4.3 10X 4.2 3.4 3.8 4.4 2.4 2.8 15X 3.9 2.7 3.3 4.5 1.92.2

The best performing benzopyrylium dye was V03-07005, with two PEG₄ andtwo sulfo groups, then, V17-03019. All benzopyrylium dyes quenched athigh molar excesses, V08-16072 and V13-06190 gradually quenched from the2.5× molar excess condition. Alexa Fluor 680 began quenching at molarexcesses greater than 10×. Due to the strong non-specific binding athigher molar excesses, Alexa-680 showed decreasing signal to background.

The excitation/emission spectra (Ex/Em spectra) were within +/−10 nmcompared to DyLight 680 NHS ester. Excitation of V03-07005, V08-16072,V13-06190, and V1703019 NHS were shifted from 690 nm in ethanol to about675 nm in PBS.

At low molar excesses, e.g., less than 10×, labeling efficiency of GAMand GAR was similar for all benzopyrylium dyes including non-pegylatedDyLight 680.

Conjugates made with the pegylated benzopyrylium dyes showed brighterstaining than the non-pegylated DyLight 680, and all stained with higherintensity than Alexa-Fluor 680 conjugates.

The pegylated benzopyrylium dyes performed best at low molar excesses asopposed to the benzocyanine dye V08-15173, which showed increasingstaining with higher amount of dye. The best performing benzopyryliumdye was V03-07005, with two PEG₄ groups on same nitrogen and two sulfogroups, followed by V17-03019, with one PEG₄ group and three sulfogroups. Absence or low non-specific binding was observed with allbenzopyrylium dyes. Non-specific binding was observed with Alexa Fluor680 at higher molar excess of 10× and 15×. All the benzopyryliumdye-conjugates quenched at high molar excesses, e.g., greater than 10×,V08-16072 and V13-06190 showed gradual quenching from 2.5× molar excesscondition.

EXAMPLE 5

In vivo imaging of inventive and commercial compounds was performed, andbiodistribution, clearance and cytotoxicity were assessed.

One hundred μL of 1 mg/mL hydrolyzed dye solution was intravenously (IV)injected in the retro orbital plexus. Animals were imaged on aCarestream MSFX using excitation of 690 nm and emission of 750 nm for 60seconds with no binning at 140 mm FOV. Images were taken beforeinjection, and at 0 hour, 3 hour, 6 hour, 12 hour, and 24 hour postinjection. After the final time point, animals were sacrificed andtissues collected for ex vivo imaging. Heart, liver, spleen, lungs, andkidney were gathered from one mouse from each cohort, and fixed andstained using hemotaxylin and eosin (H&E). Colorimetric images wereacquired at 20× on a Nikon 90i microscope (excluding DyLight 700B1).

682 Compound 2/3 (V03-07005) was used in in vivo imaging as describedabove. Biodistribution is shown in FIG. 20 at the time points shown.Ex-vivo accumulation in various organs (blood, heart, lungs, liver,kidney, spleen, GI tract, and muscle) is shown in FIG. 21. Clearance isshown in FIG. 22. Tissue toxicity of 682 Compound 2/3 (V03-07005) wasdetermined for heart (FIG. 23A), kidney (FIG. 23B), liver (FIG. 23C),lung (FIG. 23D), and spleen (FIG. 23E).

DyLight 680 was used in in vivo imaging as described above.Biodistribution is shown in FIG. FIG. 24 at the time points shown.Ex-vivo accumulation in various organs (blood, heart, lungs, liver,kidney/spleen, GI tract, and muscle) is shown in FIG. 25. Clearance isshown in FIG. 26. Tissue toxicity of DyLight 680 was determined forheart (FIG. 27A), kidney (FIG. 27B), liver (FIG. 27C), lung (FIG. 27D),and spleen (FIG. 27E).

DyLight 680 displayed fast optical clearance profile with no histologicobservations of tissue damage. Clearance pathways (renal, hepatic-GI)were visualized from in vivo data and ex vivo images.

DyLight 700B1 showed rapid hepatic uptake, followed by GI clearance. Inone embodiment, such an inventive compound is used similarly toGastrosense (Perkin Elmer). In one embodiment, such an inventivecompound is made mutlimodal by incorporating both fluorescent and X-rayopaque elements.

EXAMPLE 6

Near infra red (NIR) fluorophores are commonly used in cell-based assaysor in vivo imaging applications. They can be useful in specific imagingor assay application based on the dye's characteristic excitation andemission spectra properties, or relative hydrophylicity/hydrophobicityattributes. In general, dyes that contain a greater number of negativecharges display high water solubility and clearance, while morehydrophobic dyes often clear by a hepatic pathway. In one embodiment,the dye is conjugated to primary and secondary antibodies that are thenused for fluorescence immunostaining in various cell types. In oneembodiment, the dye is conjugated to a targeting moiety such as aprotein or peptide, e.g., the integrin binding peptide RGD, or acarbohydrate, e.g., 2-deoxyglucose. In addition, the dye is tested in invivo for optical biodistribution and clearance in nude mice. For in vivostudies, the mice receive an IV injection of dye by the retro orbitalplexus, and are imaged on a Carestream MSFX imager (690 nmexcitation/750 nm emission) before injection and at 0 h, 3 h, 6 h, 12 h,and 24 h post injection. After the final time point, animals aresacrificed and organs are collected for ex vivo imaging. Toxicity of thedye is evaluated by histological analysis of the dissected tissuesection.

The data show that dye conjugated to an antibody at low molar excess hashigh labeling efficiency, resulting in high fluorescence intensity, goodspecificity, high signal-to-background ratio, and photostability incellular imaging. Dye biodistribution and clearance shows rapidclearance through kidney and/or gastrointestinal tract. The NIR dye isan excellent tool for imaging through tissues to circumvent endogenousfluorescent biomolecule interference or quenching.

The embodiments shown and described in the specification are onlyspecific embodiments of inventors who are skilled in the art and are notlimiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

What is claimed is:
 1. A compound according to any of general formula Ia

wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is the same ordifferent and is independently selected from H, SO₃, Z, L-Z, a PEG groupP-L-Z where P is an ethylene glycol group, a diethylene glycol group, ora polyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L-SO₂NH—P-L-Z, a caboxamide group -L-CONH—P-L-Z, hydrogen,alkyl-, tert-alkyl, aryl-, carboxyaryl-, dicarboxyaryl, heteroaryl-,cycloalkyl-, heterocycloalkyl-, alkyloxy-, alkylmercapto- with alkyl andcycloalkyl including olefin linkage residues, aryloxy-, arylmercapto-,heteroaryloxy-, heteroarylmercapto-, hydroxy-, nitro-, a carboxylicacid, an amino group, or cyano residues; where L is a divalent linear(—(CH₂)_(t)—, t=0 to 15), branched, or cyclic alkane group that can besubstituted by at least one atom of oxygen, nitrogen, substitutednitrogen, and/or sulfur; where Z is H, CH₃, alkyl group, sulfoalkyl,heteroalkyl group, NH₂, —COO⁻, —COOH, —COSH, CO—NH—NH₂, —COF, —COCl,—COBr, —COI, —COO-Su (succinimidyl/sulfosuccinimidyl), —COO-STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO-Su, —CONR′-L-COO-STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, or —CONR′-L-NH—CO—CH₂—I; each of R′ and R″ isselected from H, aliphatic group, or heteroaliphatic group, and thebiomolecule is a protein, peptide, antibody, nucleotide,oligonucleotide, biotin, or hapten; X is selected from —OH, —SH, —NH₂,—NH—NH₂, —F, —Cl, —Br, I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl),—O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, or—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; each of R¹⁰, R¹³, and R¹⁴ is the same or different and isindependently selected from aliphatic, heteroaliphatic, sulfoalkylgroup, carboxyalkyl group, heteroaliphatic with terminal SO₃, Z, L-Z,PEG group P-L-Z where P is an ethylene glycol group, a diethylene glycolgroup, or a polyethylene glycol group where the polyethylene glycolgroup is (CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, asulfonamide group -L-SO₂NH—P-L-Z, or a caboxamide group -L-CONH—P-L-Z;each of R⁷ and R⁹ is the same or different and is independentlyhydrogen, aliphatic group, heteroaliphatic group, or PEG group P-L-Zwhere P is selected from an ethylene glycol group, a diethylene glycolgroup, or a polyethylene glycol group where the polyethylene glycolgroup is (CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive; or R7and R9 together form a cyclic structure where R3 and R4 are joined usinga divalent structural element selected from —(CH₂)_(q)—,—(CH₂)_(q)O(CH₂)_(q′)—, —(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—,—OCH═CH— where each of q and q′ is the same or different and is ainteger from 1 to 6 inclusive; and R⁸ is selected from hydrogen, alkyl,sulfoalkyl, fluorine, chlorine, bromine, and PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, or apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive; each of R¹ andR², R² and R³, R³ and R⁴, R⁵ and R⁶, R⁵ and R⁸, R⁹ and R¹⁰, R¹¹ and R¹²,or R¹² and R¹³ may form one or more aliphatic, heteroaliphatic oraromatic rings, and where the resultant ring(s) is optionallysubstituted by at least one alkyl-, sulfoalkyl, tert-alkyl, aryl-,carboxyaryl-, dicarboxyaryl, heteroaryl-, cycloalkyl-,heterocycloalkyl-, alkyloxy-, alkylmercapto- with alkyl and cycloalkylincluding olefin linkage residues, aryloxy-, arylmercapto-,heteroaryloxy-, heteroarylmercapto-, hydroxy-, nitro-, sulfonic acid, acarboxylic acid, an amino group, or cyano residues; at least one ofR¹-R¹⁴ optionally contains in addition to at least one PEG an additionalsolubilizing, ionizing, or ionized substituent selected from SO₃ ⁻, PO₃²⁻, CO₂H, OH, NR₃ ⁺, cyclodextrins or sugars providing hydrophiliccharacteristics; the substituents optionally linked to chromophore by analiphatic or heteroaliphatic or cyclical spacer; Kat is a number of Na⁺,K⁺, Ca²⁺, ammonia, or other cation(s) needed to compensate the negativecharge(s); n is 0, 1, 2, or 3; o is an integer from 0 to 12 inclusive;and p is an integer from 1 to 6 inclusive.
 2. The compound of claim 1wherein when the compound is according to general formula Ia or IIa eachof R2 and R3 is independently selected from sulfoalkyl or a PEG groupP-L-Z; R5 is alkyl; R6 is t-butyl or an unsubstituted or substitutedphenyl; R11 is sulfonic acid, carboxylic acid, or an amino group; eachof R13 and R14 is independently selected from alkyl, sulfoalkyl, or aPEG group P-L-Z; and each of R1, R4, R7, R8, R9, and R12 is H; when thecompound is according to general formula Ib or IIb each of R2 and R3 isindependently selected from sulfoalkyl or a PEG group P-L-Z; R5 isalkyl; R6 is t-butyl or an unsubstituted or substituted phenyl; R11 issulfonic acid, carboxylic acid, or an amino group; each of R13 and R14is independently selected from alkyl, sulfoalkyl, or a PEG group P-L-Z;and each of R1, R4, R7, R8, R9, and R12 is H; when the compound isaccording to general formula Ic or IIc each of R2 and R3 isindependently selected from sulfoalkyl or a PEG group P-L-Z; R5 isalkyl; R6 is t-butyl or an unsubstituted or substituted phenyl; R11 issulfonic acid, carboxylic acid, or an amino group; each of R13 and R14is independently selected from alkyl, sulfoalkyl, or a PEG group P-L-Z;and each of R1, R4, R7, R8, R9, and R12 is H; and when the compound isaccording to general formula Id or IId each of R2 and R3 isindependently selected from sulfoalkyl or a PEG group P-L-Z; R5 isalkyl; R6 is t-butyl or an unsubstituted or substituted phenyl; R11 issulfonic acid, carboxylic acid, or an amino group; each of R13 and R14is independently selected from alkyl, sulfoalkyl, or a PEG group P-L-Z;and each of R1, R4, R7, R8, R9, and R12 is H.
 3. The compound of claim 2wherein at least one of R2 or R3 is sulfopropyl, R5 is methyl, and atleast one of R13 or R14 is sulfopropyl.
 4. The compound of claim 1wherein when the compound is according to general formula Ie or IIe eachof R2 and R3 is independently selected from sulfoalkyl or a PEG groupP-L-Z; R5 is alkyl; R6 is t-butyl or an unsubstituted or substitutedphenyl; R11 is sulfonic acid, carboxylic acid, or an amino group; R10 isalkyl, sulfoalkyl, or a PEG group P-L-Z; R14 is alkyl; and each of R1,R4, R7, R8, R9, and R12 is H; when the compound is according to generalformula If or IIf each of R2 and R3 is independently selected fromsulfoalkyl or a PEG group P-L-Z; R5 is alkyl; R6 is t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z;R14 is alkyl; and each of R1, R4, R7, R8, R9, and R12 is H; when thecompound is according to general formula Ig or IIg each of R2 and R3 isindependently selected from sulfoalkyl or a PEG group P-L-Z; R5 isalkyl; R6 is t-butyl or an unsubstituted or substituted phenyl; R11 issulfonic acid, carboxylic acid, or an amino group; R10 is alkyl,sulfoalkyl, or a PEG group P-L-Z; R14 is alkyl; and each of R1, R4, R7,R8, R9, and R12 is H; and when the compound is according to generalformula Ih or IIh each of R2 and R3 is independently selected fromsulfoalkyl or a PEG group P-L-Z; R5 is alkyl; R6 is t-butyl or anunsubstituted or substituted phenyl; R11 is sulfonic acid, carboxylicacid, or an amino group; R10 is alkyl, sulfoalkyl, or a PEG group P-L-Z;R14 is alkyl; and each of R1, R4, R7, R8, R9, and R12 is H.
 5. Thecompound of claim 4 wherein at least one of R2 or R3 is sulfopropyl, R5is methyl, R10 is sulfopropyl, and R14 is methyl. 6-9. (canceled) 10.The compound of claim 1 selected from the group consisting of


11. The compound of claim 1 wherein R1 and R2 form a 6-membered ring,and R3 and R4 form a 6-membered ring, resulting in the benzopyryliumportion of the compound shown below


12. The compound of claim 11 wherein R5 is alkyl; R6 is t-butyl or anunsubstituted or substituted phenyl; and each of R7, R8, and R9 is H.13. The compound of claim 1 wherein R1 and R2 form a 6-membered ring,resulting in the benzopyrylium portion of the compound shown below


14. The compound of claim 13 wherein R2 is sulfoalkyl or a PEG groupP-L-Z; R3′ is sulfonic acid, carboxylic acid, or an amino group; R5 isalkyl; R6 is t-butyl or an unsubstituted or substituted phenyl; and eachof R7, R8, and R9 is H.
 15. The compound of claim 1 further comprisingat least one of a dihydro derivative, an azo bond, or a phenyl groupdisrupting the aromatic system and resulting in a quencher.
 16. Thecompound of claim 15 selected from the group consisting of

where R1 is methoxy or a PEG group, R2 is sulfoalkyl or a PEG group, andR3 is —OH or —NH—R where R is a PEG group;

where each of R1 and R2 is independently alkyl or a PEG group, R3 issulfoalkyl or a PEG group, and R4 is —OH or —NH—R where R is a PEGgroup; and

where each of R1 and R2 is independently alkyl or a PEG group, R3 issulfoalkyl or a PEG group, and R4 is —OH or —NH—R where R is a PEGgroup.
 17. A method for in vivo imaging, the method comprisingintravenously injecting a compound of claim 1 into a living animal, andobtaining at least one image of at least a portion of the animal usingthe compound.
 18. The method of claim 17 further comprising obtainingthe image during injection, after injection, or both during and afterinjection of the compound.
 19. The method of claim 17 where the compoundis injected into a circulatory system.
 20. The method of claim 17further comprising obtaining ex vivo images of at least a portion of theanimal.
 21. The method of claim 17 wherein the compound is conjugated toa targeting molecule selected from the group consisting of a peptide, aprotein, a nucleotide, an oligonucleotide, a nucleic acid, a sugar, anenzyme substrate, an enzyme antagonist, an enzyme inhibitor, and areceptor-binding compound.
 22. A method of labeling at least onebiomolecule, the method comprising providing a composition comprising atleast one excipient and the compound of claim 1 in an effectiveconcentration to the at least one biomolecule under conditionssufficient for labeling the biomolecule with the compound.
 23. Themethod of claim 22 further comprising detecting the labeled biomoleculeby at least one of fluorescence microscopy, flow cytometry, in vivoimaging, immunoassay, hybridization, chromatographic assay,electrophoretic assay, microwell plate based assay, fluorescenceresonance energy transfer (FRET) system, high throughput screening, ormicroarray.
 24. The method of claim 22 where the biomolecule is selectedfrom a protein, antibody, enzyme, nucleoside triphosphate,oligonucleotide, biotin, hapten, cofactor, lectin, antibody bindingprotein, carotenoid, carbohydrate, hormone, neurotransmitter, growthfactors, toxin, biological cell, lipid, receptor binding drug,fluorescent proteins, organic polymer carrier material, inorganicpolymeric carrier material, and combinations thereof.
 25. A kit forlabeling at least one biomolecule in a sample, the kit comprising thecompound of claim 1, at least one excipient, and instructions for usingthe compound to label the biomolecule in a sample.